Advances in Animal Genomics [1° ed.] 0128205954, 9780128205952

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Advances in Animal Genomics

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Advances in Animal Genomics

Edited by

Sukanta Mondal Ram Lakhan Singh

Academic Press is an imprint of Elsevier 125 London Wall, London EC2Y 5AS, United Kingdom 525 B Street, Suite 1650, San Diego, CA 92101, United States 50 Hampshire Street, 5th Floor, Cambridge, MA 02139, United States The Boulevard, Langford Lane, Kidlington, Oxford OX5 1GB, United Kingdom Copyright © 2021 Elsevier Inc. All rights reserved. No part of this publication may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopying, recording, or any information storage and retrieval system, without permission in writing from the publisher. Details on how to seek permission, further information about the Publisher’s permissions policies and our arrangements with organizations such as the Copyright Clearance Center and the Copyright Licensing Agency, can be found at our website: www. elsevier.com/permissions. This book and the individual contributions contained in it are protected under copyright by the Publisher (other than as may be noted herein). Notices Knowledge and best practice in this field are constantly changing. As new research and experience broaden our understanding, changes in research methods, professional practices, or medical treatment may become necessary. Practitioners and researchers must always rely on their own experience and knowledge in evaluating and using any information, methods, compounds, or experiments described herein. In using such information or methods they should be mindful of their own safety and the safety of others, including parties for whom they have a professional responsibility. To the fullest extent of the law, neither the Publisher nor the authors, contributors, or editors, assume any liability for any injury and/or damage to persons or property as a matter of products liability, negligence or otherwise, or from any use or operation of any methods, products, instructions, or ideas contained in the material herein. Library of Congress Cataloging-in-Publication Data A catalog record for this book is available from the Library of Congress British Library Cataloguing-in-Publication Data A catalogue record for this book is available from the British Library ISBN: 978-0-12-820595-2 For information on all Academic Press publications visit our website at https://www.elsevier.com/books-and-journals

Publisher: Charlotte Cockle Acquisitions Editor: Patricia Osborn Editorial Project Manager: Liz Heijkoop Production Project Manager: Debasish Ghosh Cover Designer: Miles Hitchen Typeset by TNQ Technologies

Dedicated to our beloved daughters Anoushka (Daughter of Mrs. Shrabanti Mondal and Dr. Sukanta Mondal), for making everything worthwhile and cheerfully tolerating the many hours of absence involved in writing this book and Suneha (Daughter of Dr. (Mrs.) Uma Lakhan Singh and Dr. Ram Lakhan Singh), who always functions as our lifeline and remains concerned for every single bit related to us

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Contents Contributors Preface Acknowledgments

XV XVII XXI

Pradeep Kumar Singh, Pankaj Singh, Rajat Pratap Singh and Ram Lakhan Singh

1. Introduction Pankaj Singh, Sukanta Mondal and Ram Lakhan Singh 1.1 Introduction 1.2 Branches of animal genomics 1.2.1 Structural genomics 1.2.2 Functional genomics 1.2.3 Epigenomics 1.2.4 Metagenomics 1.2.5 Pharmacogenomics 1.3 Genetic markers used in animal genomics 1.3.1 Restriction fragment length polymorphism (RFLP) 1.3.2 Random amplified polymorphic DNA (RAPD) 1.3.3 Microsatellites 1.3.4 Single nucleotide polymorphism (SNP) 1.4 Techniques used in creating transgenic animals 1.4.1 Microinjection 1.4.2 Somatic cell nuclear transfer (SCNT) 1.4.3 Artificial chromosome transfer 1.4.4 Embryonic stem (ES) cell-based cloning and transgenesis 1.4.5 Viral vector-mediated DNA transfer 1.5 Application of animal genomics 1.5.1 Livestock breeding industry 1.5.2 Transgenic animal 1.5.3 Gene therapy 1.5.4 Superovulation 1.5.5 Improving hair and fiber 1.5.6 Disease resistant animals 1.5.7 Nutritious food 1.6 Conclusion References Further reading

2. From gene to genomics: tools for improvement of animals

1 2 2 2 2 3 3 4 4 5 5 5 5 5 7 7 7 7 8 8 8 9 9 10 10 10 10 11 12

2.1 Introduction 2.2 Genes 2.2.1 Chromosome structure and organization 2.2.2 Gene structure and organization 2.3 Genome 2.3.1 Anatomy of the eukaryotic genome 2.3.2 Sequencing genomes 2.3.3 The methodology for DNA sequencing 2.3.4 The Human Genome Projects 2.3.5 Genomic libraries 2.3.6 cDNA libraries 2.4 Genomics 2.4.1 Types of genomics 2.5 Evolution of animal genomics 2.5.1 Mapping genomes 2.5.2 Regulation of gene expression 2.6 Role of genomics in animal improvement 2.7 Conclusions References

13 13 14 15 17 17 18 19 20 20 21 22 22 25 26 27 29 29 29

3. Stem cells: a potential regenerative medicine for treatment of diseases Dhruba Malakar, Hruda Nanda Malik, Dinesh Kumar, Sikander Saini, Vishal Sharma, Samreen Fatima, Kamlesh Kumari Bajwa and Satish Kumar 3.1 Introduction 3.1.1 Totipotent stem cells 3.1.2 Pluripotent stem cells 3.1.3 Multipotent stem cells 3.1.4 Oligopotent stem cells 3.1.5 Unipotent stem cells 3.2 History of stem cells 3.2.1 Types of stem cells

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3.3 Materials and methods 3.3.1 Cryopreservation of mesenchymal stem cells for a long time for further use 3.3.2 Characterization of adipose tissuederived mesenchymal stem cells 3.3.3 Confirmation for the presence of MSCs on wound areas of treated animals 3.3.4 Isolation of ovarian surface epithelium cells for generation of oocytes 3.3.5 Characterization of OSE-derived primordial germ cell-like structure 3.4 Applications of embryonic and adult stem cells 3.4.1 Study of diseases and how they develop 3.4.2 Stem cells: a model for screening, discovery, and development of drugs 3.4.3 Transgenic animal production 3.4.4 Therapeutic cloning 3.4.5 Regenerative medicine 3.5 Current clinical applications of adult mesenchymal stem cells in regenerative medicine 3.5.1 Treatment of massive wounds of animals 3.5.2 Mastitis treatment 3.5.3 Metritis and endometritis 3.5.4 Bone fracture and orthopedic defects 3.5.5 Spinal cord injury 3.5.6 Treatment of dogs 3.5.7 Blood stem cell transplantation 3.5.8 Burn therapy 3.5.9 Corneal regeneration 3.5.10 Immunomodulatory disease treatments 3.5.11 Neurodegenerative diseases 3.5.12 Liver diseases 3.5.13 Cardiac related diseases 3.5.14 Treatment of diabetes with MSCs 3.5.15 Treatment of cancer with MSCs 3.6 Challenges of stem cells 3.6.1 Stem cells in reproduction and infertility 3.6.2 Testis xenografting 3.6.3 Spermatogonial stem cell transplantation

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37 37 37 38 38 38 39 39

39 41 41 42 43 43 43 43 43 44 44 44 44 45 45 45 45 45 46 46

3.6.4 Spermatogonial stem cells as a source for fertility restoration 3.6.5 Generation of oocytes from ovarian surface epithelium for regenerative medicine 3.7 Conclusion References

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4. Alternative transcriptome analysis to build the genome-phenome bridges in animals Xuelei Han Songlei Xue, Amy L. Zinski, Shane Carrion, Jennifer J. Michal and Zhihua Jiang 4.1 Introduction 4.2 Modern sequencing platforms and transcriptome profiling strategies 4.2.1 High-throughput sequencing technologies 4.2.2 RNA sequencing 4.2.3 50 -end sequencing 4.2.4 30 -end sequencing 4.2.5 Isoform sequencing 4.2.6 Single-cell RNA sequencing 4.3 Genome-wide profiling of ATS and APA sites 4.3.1 WTSS-seq and WTTS-seq design 4.3.2 Characterization of ATS and APA sites 4.3.3 WTSS-seq and WTTS-seq: mutual validation 4.3.4 Advantages of WTTS-seq over RNA-seq 4.4 Genome to phenome via alternative transcriptome 4.4.1 Alternative transcriptome: bridges between genome and phenome 4.4.2 Alternative transcriptome: interactions with gene biotypes 4.4.3 Alternative transcriptome: alteration under gene knockouts 4.4.4 Alternative transcriptome: responses to a high-fat diet 4.4.5 Alternative transcriptome: the future of genome biology Acknowledgments References

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5. RNA sequencing: a revolutionary tool for transcriptomics Minal Garg 5.1 Introduction 5.2 Transcriptional landscape: regulatory RNAs 5.3 Transcriptome sequencing 5.3.1 RNA isolation, reverse transcription and library preparation 5.3.2 Sequencing platforms 5.3.3 Analysis of the transcriptional landscape 5.3.4 Differential gene expression analysis 5.3.5 Alternatively-spliced transcript analysis 5.3.6 Allele specific expression 5.3.7 Fused gene analysis 5.3.8 Small RNA analysis 5.3.9 Expression quantitative trait loci analysis 5.4 Future perspectives References

61 61 63 63 65 66 68 68 69 69 69 70 70 71

6. Targeted genome editing: a new era in molecular biology 75 77 78 78 79 79 81 81 82 82 83 84 85 85 86 87

7. RNAi for livestock improvement Uzma Noor Shah, Shanmugapriya Gnanasekaran, Sukanta Mondal, I.J. Reddy, S. Nandi, P.S.P. Gupta and D.N. Das 7.1 Introduction 7.2 History behind RNAi

92 92 92 92 93 93 94 94 94 95 97 100 100 101 101 101 101 102 106

8. Microbial metagenomics: potential and challenges

Devlina Ghosh, Alok Kumar and Neeraj Sinha 6.1 Introduction 6.2 Homologous recombination 6.2.1 Scientific/clinical significance 6.3 Endonucleases/zinc-finger nucleases 6.3.1 Scientific/clinical significance 6.4 Transcription activator-like effector nucleases (TALENS) 6.4.1 Scientific/clinical significance 6.5 CRISPR-Cas9 6.5.1 Origin of CRISPR-Cas9 6.5.2 Underlying mechanism 6.6 Scientific advantage/applications 6.7 Clinical aspect 6.7.1 Limitations 6.8 Ethical concerns 6.9 Conclusion References

7.3 Versatility of RNA molecule 7.4 Mechanism of RNAi 7.5 Pathways of RNA silencing 7.5.1 Posttranscriptional gene silencing (PTGS) 7.5.2 Transcriptional gene silencing (TGS) 7.6 The miRNA pathway 7.7 piRNA 7.8 Transgenesis in livestock improvement 7.9 RNAi in livestock 7.10 Transgenic expression of RNAi-inducing molecules 7.11 Applications of RNAi in livestock 7.12 RNAi in functional genomics 7.12.1 RNA therapeutics 7.12.2 Molecular insights into stem cell biology and genetic engineering 7.12.3 Toward environmentally friendly farm animals 7.13 Challenges 7.14 Conclusions References Further reading

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Shikha, Shailja Singh and Shiv Shankar 8.1 Introduction 8.2 Metagenome and metagenomics 8.2.1 Habitat selection 8.2.2 Sampling 8.2.3 Macromolecule recovery 8.3 Next-generation sequencing (NGS) to explore microbial communities 8.3.1 Pyrosequencing 8.3.2 Reconstructing the genomic content of the microbial community from NGS data 8.3.3 Amplicon sequencing analyses 8.3.4 Shotgun metagenomics 8.4 Bioprospecting of metagenomes 8.4.1 Sequence-based analyses 8.4.2 Function-based analyses 8.5 Applications of metagenomics 8.5.1 Biocatalysts and metagenomics 8.5.2 Metagenomics and pharmaceuticals 8.5.3 Metagenomics and biosurfactants 8.5.4 Metagenomics and biodegradation 8.6 Conclusions and future perspectives References

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111 111 112 113 113 114 115 115 118 119 119 119 120

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9. Molecular markers and its application in animal breeding Reshma Raj S and D.N. Das 9.1 Introduction 9.2 Quantitative and molecular genetics 9.3 Molecular markers 9.3.1 Restriction fragment length polymorphism (RFLP) 9.3.2 Random amplified polymorphic DNA (RAPD) 9.3.3 Amplified fragment length polymorphism (AFLP) 9.3.4 Microsatellites 9.3.5 Minisatellites 9.3.6 Single nucleotide polymorphisms (SNPs) 9.3.7 Allozyme markers 9.3.8 Mitochondrial DNA (mtDNA) 9.3.9 DNA barcoding markers 9.4 Marker assisted selection (MAS) 9.4.1 Applications of MAS 9.5 Conclusion References Further reading

123 123 124 125 127 128 130 131 131 132 132 134 135 135 136 136 139

10. Genomic selection: a molecular tool for genetic improvement in livestock D.N. Das, T. Karuthadurai and Shanmugapriya Gnanasekaran 10.1 Introduction 10.2 Conventional selection 10.3 Selection - the major tool for genetic improvement 10.4 Principles of selection 10.5 Natural selection 10.6 Artificial selection 10.7 Selection for additive gene action 10.8 Selection for multiple alleles 10.9 Selection for epistasis 10.10 Selection intensity 10.11 Generation interval 10.12 Selection accuracy 10.13 Phenotypic value 10.14 Breeding value 10.15 Population mean 10.16 Average effect 10.17 Dominance deviation 10.18 Genetic control on production traits 10.19 Genetic control on reproductive traits

141 141 142 142 142 143 143 143 143 144 144 144 144 145 145 145 145 146 146

10.20 Genetic control on embryonic mortality in dairy cows 10.21 Marker assisted selection 10.22 MAS versus conventional selection 10.23 Whole-genome selection 10.24 Principle of genomic selection 10.25 Estimation of genomic breeding value 10.26 Factors influencing genomic selection 10.27 Present status of genomic selection in livestock breeding schemes 10.28 Prospects of genomic selection in cattle and buffaloes in India 10.29 Genetic gain by genomic selection 10.30 Methods of genomic selection 10.31 Advantage of genomic selection over conventional breeding 10.32 Reabilities (r2v) of EBV and regression coefficient (REG) of corrected phenotypic values 10.33 Genomic evaluations in developing versus developed countries 10.34 Genomic selection in developed countries 10.35 Genome-wide signatures for selection using molecular genomic tools 10.35.1 SNP Chip 10.36 Functional genomics in fertility traits 10.37 Approaches for developing disease tolerant livestock 10.38 Candidate genes for disease resistance for milk production 10.39 Production of disease-resistant genetically modified livestock 10.39.1 Dominant negative proteins 10.39.2 Ribonucleic acid interference (RNAi) 10.39.3 Ribonucleic acid decoys 10.39.4 Animal pharming 10.39.5 Antibodies 10.40 CRISPR 10.41 RNA editing 10.42 Disease treatment 10.43 Conclusion References

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152 152 156 156 156 157 158 158 158 159 159 159 160 160 160 160 161 161 161

11. Gene therapy Deepti Saini 11.1 11.2 11.3 11.4

Genes Gene therapy Use of gene therapy Types of gene therapy: somatic and germline

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11.5 11.6 11.7 11.8 11.9

Types of vectors Techniques of gene therapy History of human gene therapy CRISPR gene editing Gene therapy in animals 11.9.1 Large animal disease models 11.9.2 Strategies, methods, and vectors for gene transfer 11.9.3 Gene therapy for the treatment of AIDS in animals 11.9.4 Brain cancer 11.9.5 The plastic bubble disease 11.9.6 Cure for blindness 11.10 Some other potential uses of gene therapy 11.11 Safety issues of gene therapy 11.11.1 Ethical and moral concerns surrounding gene therapy 11.12 Conclusion References

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12. Nanobiotechnology in animal production and health Ravindra Pratap Singh and Kshitij R.B. Singh 12.1 Introduction 12.2 Quantum dot nanoparticles 12.3 Carbon-based nanoparticles 12.4 Dendrimers nanoparticles 12.5 Liposomes nanoparticles 12.6 Metal and metal oxides nanoparticles 12.7 Polymeric nanoparticles 12.8 Conclusions Acknowledgment References Further reading

185 186 186 187 189 190 191 192 192 192 198

13. Cell signaling and apoptosis in animals M. Naveen Kumar, Shivaleela Biradar and R.L. Babu 13.1 Introduction 13.2 Cell signaling in animals 13.3 Classification of cell signaling in animal cells 13.3.1 Autocrine 13.3.2 Paracrine 13.3.3 Endocrine 13.3.4 Signaling through direct contact

199 199 199 199 200 200 200

13.4 Signaling receptors 13.4.1 Intracellular signaling receptors 13.4.2 Cell- surface signaling receptors 13.5 Second messengers in animal cell signaling 13.6 Pathways of cell signaling 13.7 Computational mapping of animal cell signaling 13.8 Apoptosis 13.9 Classification of cell death in animal cells 13.9.1 Autophagy 13.9.2 Necrosis 13.10 Cellular and biochemical feature of apoptotic cells 13.11 Apoptosis: proteins and signaling pathways 13.12 Regulatory mechanism of apoptosis in animal cells 13.13 Apoptosis deregulation and diseases 13.14 Methods of apoptosis detection 13.15 Conclusion References

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14. Molecular Network for Management of Neurodegenerative Diseases and their Translational Importance using Animal Biotechnology as a Tool in Preclinical Studies Nibedita Naha Abbreviations 14.1 Introduction 14.2 Pathogenesis and Molecular Mapping of Neurodegenerative Diseases 14.2.1 Alzheimer’s Disease (AD) 14.2.2 Parkinson’s Disease (PD) 14.2.3 Huntington’s Disease (HD) 14.2.4 Amylotrophic Lateral Sclerosis (ALS) 14.3 Drug Targets of Protein Aggregates in Neurodegenerative Diseases with Translational Impacts 14.4 Conclusion and Future Direction of Research Acknowledgment Conflict of Interest References

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15. Issues and policies in animal genomics Ramanuj Banerjee and Sukanta Mondal 15.1 Introduction 15.1.1 Genomic technologies in animal husbandry 15.2 Global (transcontinental) scenario in transgenic animal research: issues and policies 15.2.1 North America 15.2.2 South America 15.2.3 Australia/Oceania 15.2.4 Europe 15.2.5 Asia 15.2.6 Africa 15.2.7 Antarctica 15.3 Genomics vis-a-vis Indian policy and regulations: current deliberations 15.3.1 Scope of guidelines 15.3.2 Classification of pathogenic microorganisms 15.3.3 Containment 15.4 Mechanism of implementation of biosafety guidelines in India 15.4.1 Recombinant DNA Advisory Committee (RDAC) 15.4.2 Institutional Biosafety Committee (IBSC) 15.4.3 Review Committee on Genetic Manipulation (RCGM) 15.4.4 Genetic Engineering Approval Committee (GEAC) 15.4.5 State Biotechnology Coordination Committee (SBCC) 15.4.6 District Level Committee (DLC) 15.5 Assessment of environmental risk 15.5.1 Mechanisms by which the environment may be exposed to GMO hazards 15.6 Capacity to survive, establish and disseminate 15.7 Hazards associated with the inserted gene/element 15.8 Transfer of harmful sequences between organisms 15.9 Phenotypic and genetic stability 15.10 Risk assessment for human health 15.11 Mechanisms by which the GMO could be a risk to human health 15.12 Control measures to protect human health 15.12.1 Biosafety Level (BSL) facilities 15.12.2 Different Bio Safety Level nomenclatures

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15.13 Animal Bio Safety Level (ABSL) facilities 15.13.1 Operational guide for ABSL facilities 15.13.2 Types of Animal Biosafety Level facilities 15.14 Approvals and prohibitions 15.15 Conclusion Disclaimer Conflict of Interests Acknowledgments References Further reading

250 250 250 252 252 255 255 255 255 256

16. Silkworm genomics: current status and limitations Manjunatha H. Boregowda 16.1 Introduction 16.2 Genomic basis of the demographic history of the domesticated silkworm, Bombyx mori 16.3 Cytogenetics of the silkworm, Bombyx mori 16.4 Silkworm genomics 16.5 Silkworm genome programs 16.6 Silkworm genome sequence 16.6.1 Phase I: draft genome sequence 16.7 Phase II: integrated genome sequence (http://silkworm.genomics.org.cn/) 16.8 Integration method 16.8.1 Integrated genome assembly and its characteristics 16.8.2 De novo analysis for genome organization and gene count 16.8.3 Limitations in the integrated genome assembly 16.8.4 Phase III: high-quality new genome sequence and assembly 16.9 Genome sequence of domesticated and wild silkworm strains 16.10 Repetitive/transposable elements in the silkworm genome 16.11 Mapping silkworm genome 16.11.1 The linkage map 16.12 Genetic and molecular linkage maps 16.13 Limitations 16.13.1 Physical map by bacterial artificial chromosome (BAC), and fluorescence in-situ hybridization (FISH) 16.13.2 Strategies for construction of a physical map 16.13.3 Limitations in the silkworm genome mapping

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16.14 Silkworm genome resources 16.14.1 Complimentary DNA libraries (cDNA) 16.14.2 Expressed sequence tags (EST) 16.14.3 Single nucleotide polymorphisms (SNP) 16.14.4 Sequence-tagged sites (STS) 16.15 Microsatellites 16.16 Silkworm genome database and characteristics 16.16.1 KAIKObase (http:// sgp.dna.affrc.go.jp/KAIKObase/) 16.16.2 SilkDB (http://www.silkdb.org) 16.17 Limitations in the application of genome data for the improvement of silkworm strains References Web references

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17. Deciphering the animal genomics using bioinformatics approaches Talambedu Usha, Prachurjya Panda, Arvind Kumar Goyal, Shivani Sukhralia, Sarah Afreen, H.P. Prashanth Kumar, Dhivya Shanmugarajan and Sushil Kumar Middha 17.1 Introduction 17.1.1 Need for bioinformatics in animal genomics 17.1.2 Application of animal genomics 17.2 Genomic-bioinformatics processes 17.2.1 DNA sequencing 17.2.2 Alignment 17.2.3 Genome assembly 17.2.4 Annotation 17.3 Technologies to assess gene expression 17.3.1 Differential display 17.3.2 Microarrays 17.3.3 Transcriptome and RNA sequencing 17.3.4 Serial analysis of gene expression (SAGE) 17.4 Tools for genomic data manipulation 17.4.1 Database for annotation, visualization, and integrated discovery (DAVID) 17.4.2 UCSC genome bioinformatics site 17.4.3 R language and bioconductor 17.5 Animal genomes available in NCBI 17.5.1 Major genomes in aquaculture

281 281 282 282 282 283 284 284 284 284 285 285 285 286

286 286 286 287 287

17.6 Databases/major genomes available in animal genomics 17.7 Popular genomes of domestic animals 17.7.1 Buffalo 17.7.2 Goat 17.7.3 Sheep 17.7.4 Cow 17.8 India on world genomes map in animal genomics 17.9 Future prospects Acknowledgment References

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18. DNA barcoding: nucleotide signature for identification and authentication of livestock Kunal Ankola, LikhithGowda Mahadevegowda, Tomas Melichar and Manjunatha H. Boregowda 18.1 Introduction 18.2 DNA barcoding as a technique to generate nucleotide signature 18.3 DNA barcoding in livestock management 18.4 The advent of DNA barcoding 18.5 Nucleotide signature and barcode 18.6 Types of DNA barcoding 18.6.1 Meta-barcoding 18.6.2 Mini-barcoding 18.7 Methods involved in DNA barcoding 18.7.1 Extraction and purification of DNA from the samples 18.7.2 The genetic markers to barcode 18.7.3 Barcode and Polymerase Chain Reaction 18.7.4 Nucleotide/molecular signature 18.7.5 Reference libraries and databases 18.7.6 Barcoding sequence analysis 18.8 Applications of DNA barcode 18.8.1 Identification of new species 18.8.2 Advantage of DNA barcoding in livestock management 18.8.3 Cryptic species demarcation 18.8.4 Barcode-based diet analysis in animals 18.9 DNA barcode and intellectual property right (IPR) Acknowledgments References

Index

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Contributors Sarah Afreen, DBT-BIF Facility, Department of Biotechnology, Maharani Lakshmi Ammanni College for Women, Bengaluru, Karnataka, India Kunal Ankola, Department of Studies in Sericulture Science, University of Mysore, Mysore, Karnataka, India R.L. Babu, Department of Bioinformatics and Biotechnology, Karnataka State Akkamahadevi Women’s University, Jnanashakthi Campus, Vijayapura, Karnataka, India Kamlesh Kumari Bajwa, Animal Biotechnology Centre, National Dairy Research Institute, Karnal, Haryana, India Ramanuj Banerjee, Department of Scientific & Industrial Research, Ministry of Science & Technology, Government of India, New Delhi, Delhi, India Shivaleela Biradar, Department of Bioinformatics and Biotechnology, Karnataka State Akkamahadevi Women’s University, Jnanashakthi Campus, Vijayapura, Karnataka, India Manjunatha H. Boregowda, Department of Studies in Sericulture Science, University of Mysore, Mysore, Karnataka, India

Shanmugapriya Gnanasekaran, Genetics Laboratory, Dairy Production Section, ICAR-National Dairy Research Institute (SRS), Southern Regional Station, Bangalore, Karnataka, India Arvind Kumar Goyal, Centre for Bamboo Studies, Department of Biotechnology, Bodoland University, Kokrajhar, Assam, India P.S.P. Gupta, ICAR-National Institute of Animal Nutrition and Physiology, Bangalore, Karnataka, India Zhihua Jiang, Department of Animal Sciences, Washington State University, Pullman, WA, United States T. Karuthadurai, Genetics Laboratory, Dairy Production Section, ICAR-National Dairy Research Institute (SRS), Southern Regional Station, Bangalore, Karnataka, India Alok Kumar, Department of Molecular Medicine & Biotechnology, Sanjay Gandhi Postgraduate Institute of Medical Sciences, Lucknow, Uttar Pradesh, India Dinesh Kumar, Animal Biotechnology Centre, National Dairy Research Institute, Karnal, Haryana, India Satish Kumar, Animal Biotechnology Centre, National Dairy Research Institute, Karnal, Haryana, India

Shane Carrion, Department of Animal Sciences, Washington State University, Pullman, WA, United States

LikhithGowda Mahadevegowda, Department of Studies in Sericulture Science, University of Mysore, Mysore, Karnataka, India

D.N. Das, Genetics Laboratory, Dairy Production Section, ICAR-National Dairy Research Institute (SRS), Southern Regional Station, Bangalore, Karnataka, India

Dhruba Malakar, Animal Biotechnology Centre, National Dairy Research Institute, Karnal, Haryana, India

Samreen Fatima, Animal Biotechnology Centre, National Dairy Research Institute, Karnal, Haryana, India Minal Garg, Department of Biochemistry, University of Lucknow, Lucknow, Uttar Pradesh, India Devlina Ghosh, Centre of Biomedical Research, SGPGIMS-Campus, Lucknow, Uttar Pradesh, India; Amity Institute of Biotechnology, Amity University, Lucknow Campus, Gomti Nagar Extension, Lucknow, Uttar Pradesh, India

Hruda Nanda Malik, Animal Biotechnology Centre, National Dairy Research Institute, Karnal, Haryana, India Tomas Melichar, Sphingidae Museum, Orlov, Czech Republic Jennifer J. Michal, Department of Animal Sciences, Washington State University, Pullman, WA, United States Sushil Kumar Middha, DBT-BIF Facility, Department of Biotechnology, Maharani Lakshmi Ammanni College for Women, Bengaluru, Karnataka, India

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Sukanta Mondal, Principal Scientist, ICAR-National Institute of Animal Nutrition and Physiology, Adugodi, Bangalore, Karnataka, India

Shikha, Department of Environmental Science, Babasaheb Bhimrao Ambedkar University, Lucknow, Uttar Pradesh, India

Nibedita Naha, Biochemistry Division, ICMReNational Institute of Occupational Health (NIOH), Ahmedabad, Gujarat, India

Pankaj Singh, Department of Biotechnology, Dr. Rammanohar Lohia Avadh University, Ayodhya, Uttar Pradesh, India

S. Nandi, ICAR-National Institute of Animal Nutrition and Physiology, Bangalore, Karnataka, India

Pradeep Kumar Singh, Department of Biochemistry, Dr. Rammanohar Lohia Avadh University, Ayodhya, Uttar Pradesh, India

M. Naveen Kumar, Department of Biotechnology and Genetics, M.S. Ramaiah College of Arts, Science and Commerce, Bengaluru, Karnataka, India Prachurjya Panda, DBT-BIF Facility, Department of Biotechnology, Maharani Lakshmi Ammanni College for Women, Bengaluru, Karnataka, India H.P. Prashanth Kumar, Department of Biotechnology, Sapthagiri College of Engineering, Bengaluru, Karnataka, India I.J. Reddy, ICAR-National Institute of Animal Nutrition and Physiology, Bangalore, Karnataka, India Reshma Raj S, ICAR-National Dairy Research Institute, Bangalore, Karnataka, India Deepti Saini, Protein Design Pvt. Ltd, SID, Indian Institute of Science, Bangalore, Karnataka, India Sikander Saini, Animal Biotechnology Centre, National Dairy Research Institute, Karnal, Haryana, India Uzma Noor Shah, ICAR-National Institute of Animal Nutrition and Physiology, Bangalore, Karnataka, India Shiv Shankar, Department of Environmental Science, School of Vocational Studies and Applied Sciences, Gautam Buddha University, Greater Noida, Uttar Pradesh, India Dhivya Shanmugarajan, DBT-BIF Facility, Department of Biotechnology, Maharani Lakshmi Ammanni College for Women, Bengaluru, Karnataka, India Vishal Sharma, Animal Biotechnology Centre, National Dairy Research Institute, Karnal, Haryana, India

Ravindra Pratap Singh, Department of Biotechnology, Indira Gandhi National Tribal University, Amarkantak, Madhya Pradesh, India Kshitij R.B. Singh, Department of Biotechnology, Indira Gandhi National Tribal University, Amarkantak, Madhya Pradesh, India Shailja Singh, Department of Environmental Science, Babasaheb Bhimrao Ambedkar University, Lucknow, Uttar Pradesh, India Rajat Pratap Singh, Department of Biotechnology, Guru Ghasidas University, Bilaspur, Chhattisgarh, India Ram Lakhan Singh, Department of Biochemistry, Dr. Rammanohar Lohia Avadh University, Ayodhya, Uttar Pradesh, India; Present Address: Vice-Chancellor, Nilamber-Pitamber University, Medininagar, Palamu, Jharkhand, India Neeraj Sinha, Centre of Biomedical Research, SGPGIMSCampus, Lucknow, Uttar Pradesh, India Xuelei Han, Department of Animal Sciences, Washington State University, Pullman, WA, United States Shivani Sukhralia, DBT-BIF Facility, Department of Biotechnology, Maharani Lakshmi Ammanni College for Women, Bengaluru, Karnataka, India Talambedu Usha, Department of Biochemistry, Bangalore University, Bengaluru, Karnataka, India Songlei Xue, Department of Animal Sciences, Washington State University, Pullman, WA, United States Amy L. Zinski, Department of Animal Sciences, Washington State University, Pullman, WA, United States

Preface Globally, critical issues facing agriculture include delivery of human health care, reduction in hunger, and increasing energy supply, all in a sustainable manner with optimum animal welfare and minimal negative impact on the environment. The United Nations (UN) predicted that the world population would exceed nine billion by 2030, and improving the quality and quantity of food production is an inevitable necessity. According to the UN, this doubled food requirement must come from virtually the same land area as today. The UN Food and Agriculture Organization (FAO) further stated that 70 percent of this additional food must come from the use of new and existing agricultural technologies. The FAO has also estimated that in the same time frame, livestock production would produce nearly 20 percent of the global greenhouse gas emissions. Notwithstanding the environmental challenge, by the end of the next decade, the livestock sector is expected to provide 50 percent of global agricultural output on a value basis. Livestock plays an important role in the growth of the agricultural sector in developing economies. The role of the livestock sector is crucial to fulfilling the growing food demand, which is expected to increase by 40 percent by 2030 and would almost be doubled by 2050. The increased demand for livestock products can be met by enhancing the numbers of animals, improving feed utilization efficiency, adopting better reproductive strategies, and improving health coverage based on newer generation biotechnological vaccines and drugs. Animal biotechnology is set to become an essential tool in the effort to meet the growing global demand for meat and milk. Advances in animal biotechnology can help livestock producers increase productivity to meet future nutritional, energy, and fiber needs while maintaining the quality of life for animals used for food, fiber, work, or pleasure and decreasing the environmental impacts. The recent advent of high-throughput next-generation whole-genome and transcriptome sequencing, array-based genotyping, and modern bioinformatics approaches have enabled the production of huge genomic and transcriptomic resources globally on a genome-wide scale. The advances in genome technology plays an important role in the improvement of livestock productivity, conservation of domestic animal diversity, molecular diagnostics for animal and crop health, improved vaccines against transboundary animal diseases, etc. The developments in molecular biology and biotechnology have resulted in unlimited access to the gene pool and enhanced the pace and precision of creating gene sequencing and functional genomics to meet the challenges of food, agriculture, and animal improvement. The sequencing of the livestock genomes has led to the discovery of genome-wide Deoxyribonucleic Acid (DNA) markers, which, in turn, paved the way for the development of DNA chips enabling genomic selection in livestock. Moreover, genetic engineering has the potential to provide compelling benefits to transform public health, including improved foods, advances for human health, enhanced animal welfare, and reduced environmental impact. In recent years, tremendous progress has been made in animal genomics and molecular breeding research pertaining to the conventional and next-generation whole genome, transcriptome, and epigenome sequencing efforts, generation of huge genomic, transcriptomic and epigenomic resources, and development of modern genomicsassisted breeding approaches in diverse animal genotypes with contrasting yield and abiotic stress tolerance traits. Unfortunately, the detailed molecular mechanism and gene regulatory networks controlling such complex quantitative traits are still not well understood in livestock. Recent advances in Animal Genomics is an outstanding collection of integrated strategies involving available enormous and diverse traditional and modern genomics (structural, functional, comparative, and epigenomics) approaches/resources, and genomics-assisted breeding methods, which animal biotechnologist can adopt/utilize to dissect and decode the molecular and gene regulatory networks involved in the complex quantitative yield and stress tolerance traits in livestock. The book is particularly attractive for scientists, researchers, students, educators, and professionals in agriculture, veterinary, and biotechnology science. This book will enable them to solve the problems about sustainable development with the help of current innovative biotechnologies such as recombinant DNA technology and genetic engineering, which have tremendous potential for impacting global food security, environmental health, human and animal health, and the overall livelihood of mankind.

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The contributors to the book are internationally recognized experts in their field, and they represent reputed institutions across the globe.

Key features of the book The text of the book includes certain important features to facilitate a better understanding of the topics discussed in the chapters. A summary has been presented at the beginning of each chapter to highlight the important concepts discussed in the chapter. The contents of each chapter list the main topics included in that chapter. Tables and figures dispersed throughout the chapters enable easy understanding of the concepts discussed. The bibliography at the end of each chapter familiarizes the readers with important texts and articles cited in the book.

Organization of the book This book consists of 18 chapters that focus on current approaches and strategies for sustainable livestock production. The introduction traces the brief introduction, scope, and applications of animal genomics for sustainable livestock production. From gene to genomics: tools for improvement of animals describe the gene structure and organization, eukaryotic genome, sequencing genomes, methodology for DNA sequencing, the evolution of animal genomics, and role of genomics in animal improvement. Stem cells: a potential regenerative medicine for the treatment of diseases involves definition, history of stem cells, types of stem cells, applications of embryonic and adult stem cells, current clinical applications of adult mesenchymal stem cells in regenerative medicine, and challenges of stem cells. Alternative transcriptome analysis to build the genome-phenome bridges in animals deals with modern sequencing platforms and transcriptome profiling strategies, high-throughput sequencing technologies, genome-wide profiling of ATS and APA sites, and genome to phenome via alternative transcriptome. RNA sequencing: a revolutionary tool for transcriptomics deals with the transcriptional landscape: regulatory RNAs and its analysis, transcriptome sequencing, and future perspectives. Targeted genome editing: a new era in molecular biology highlights homologous recombination, Endonucleases/ Zinc Finger Nucleases, Transcription Activator-Like Effector Nucleases (TALENS), CRISPR-Cas9, scientific advantage/ applications, clinical aspect, limitations, and ethical concerns. RNAi for livestock improvement demonstrates history, mechanism of silencing gene expression by RNAi, transgenic expression of RNAi-inducing molecules, applications of RNAi in livestock, RNAi in functional genomics, and challenges. Microbial metagenomics: potential and challenges cover metagenome and metagenomics, Next-generation sequencing (NGS) to explore microbial communities, bioprospecting of metagenomes, applications of metagenomics, conclusions, and future perspectives. Molecular markers and its application in animal breeding focus on quantitative and molecular genetics, Molecular markers - Restriction fragment length polymorphism (RFLP), Random amplified polymorphic DNA (RAPD), Amplified fragment length polymorphism (AFLP), Microsatellites, Minisatellites, Single nucleotide polymorphisms (SNPs), Allozyme markers, Mitochondrial DNA (mtDNA), DNA Barcoding markers, Marker-assisted selection (MAS) and its application. Genomic selection: a molecular tool for genetic improvement in livestock involves conventional selection, natural selection, artificial selection, selection intensity and accuracy, genetic control on production traits and reproductive traits, genomic selection and factors influencing the genomic selection, methods of genomic selection, genomic evaluations in developing versus developed countries, genome-wide signatures for selection using molecular genomic tools, functional genomics in fertility traits, approaches for developing disease tolerant livestock, candidate genes for disease resistance for milk production, and production of disease-resistant genetically modified livestock. Gene therapy illustrates the history of gene therapy, techniques of gene therapy, use of gene therapy in animals, and other potential uses of gene therapy, as well as safety issues of gene therapy. Nanobiotechnology in animal production and health covers quantum dot nanoparticles, carbon-based nanoparticles, dendrimers nanoparticles, liposomes nanoparticles, metal and metal oxides nanoparticles, polymeric nanoparticles, etc. Cell Signaling and apoptosis in animals involves cell signaling in animals, classification of cell signaling, signaling receptors, second messengers in cell signaling, pathways of cell signaling and signal transduction, computational mapping of animal cell signaling, classification of cell death, cellular and biochemical features of apoptotic cells, proteins and signaling pathways, the regulatory mechanism of apoptosis, apoptosis deregulation, and methods of apoptosis detection.

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Molecular network for management of neurodegenerative diseases and its translational importance using animal biotechnology as a tool in preclinical studies involves pathogenesis and molecular mapping of neurodegenerative diseases -Alzheimer’s disease (AD), Parkinson’s disease (PD), Huntington’s disease (HD), and Amyotrophic lateral sclerosis (ALS), drug targets of protein aggregates in neurodegenerative diseases, with translational impacts and future direction of research. Issues and policies in animal genomics demonstrates genetic engineering technology/genomics for animal husbandry application, genetic engineering technology/genomics for animal husbandry application, genomics versus policy, regulations in India: current deliberations, mechanism of implementation of biosafety guidelines in India, risk assessment for the environment, mechanisms by which the GMO might pose a hazard to the environment, hazards associated with the inserted gene/element, risk assessment for human health, control measures needed to sufficiently protect human health, and microbiological biosafety level (BSL) facilities. Silkworm genomics: current status and limitations discuss genomic basis of the demographic history of the domesticated silkworm, Bombyx mori, cytogenetics of the silkworm, Bombyx mori, silkworm genomics, silkworm genome programs, silkworm genome sequence, draft genome sequence, integrated genome sequence, and high-quality new genome sequence and assembly, genome sequence of domesticated and wild silkworm strains, repetitive/transposable elements in the silkworm genome, mapping silkworm genome and limitations. Deciphering the animal genomics using bioinformatics approaches illustrates the need for bioinformatics in animal genomics, genomic-bioinformatics processes, technologies to assess gene expression, tools for genomic data manipulation, animal genomes available in NCBI, databases/major genomes available in animal genomics, popular genomes of domestic animals, India on world genomes map in animal genomics and future prospects. DNA barcoding covers the advent of DNA barcoding, nucleotide signature and barcoding, types of DNA barcoding methods involved in DNA barcoding, applications of DNA barcode, DNA barcoding, and intellectual property rights (IPR). Sukanta Mondal Ram Lakhan Singh

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Acknowledgments It is a pleasure to acknowledge and express our enormous debt to all the contributors who assisted materially in the preparation of this book. We are grateful to both our families, who cheerfully supported and tolerated the many hours of our absence for finishing this book project. Thanks are also due to Heijkoop Liz, Maragioglio Nancy, Osborn Patricia, Debasish Ghosh, and the entire publishing team for their patience and extra care in publishing this book. Sukanta Mondal Ram Lakhan Singh

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Chapter 1

Introduction Pankaj Singh1, Sukanta Mondal2 and Ram Lakhan Singh3, 4, * 1

Department of Biotechnology, Dr. Rammanohar Lohia Avadh University, Ayodhya, Uttar Pradesh, India; 2Principal Scientist, ICAR-National

Institute of Animal Nutrition and Physiology, Adugodi, Bangalore, Karnataka, India; 3Department of Biochemistry, Dr. Rammanohar Lohia Avadh University, Ayodhya, Uttar Pradesh, India; 4Vice-Chancellor, Nilamber-Pitamber University, Medininagar, Jharkhand, India

1.1 Introduction The study of the genome is called genomics. Genomics is the subdiscipline of genetics, which includes mapping, sequencing, and functional analysis of all genes present in an organism’s genome. The term “genome” was given by German botanist Hans Winkler by merging the words gene and chromosome. The genome includes the total number of chromosomes in ova or spermatozoa in humans or animals. Later on, Lederberg and McCray suggested that the term genome consists of a gene with the generalized suffix “ome,” which means “the entire collectivity of units,” and not some “body” from the chromosome. Genomics involves the study of entire genes of the genome at replication, transcription, and translation level at the cellular or tissue level and intergenomic interactions of the genome (Fig. 1.1). The main objectives of genomics are: l l l

Study of organization and expression of the genome as specific traits. Characterization of the complete genome rather than one gene at a time with the application of new technologies. Sequencing of mainly livestock and poultry genomes to understand the functional genomics.

The discovery of new techniques and technologies in the field of omic sciences opens new concepts about the genomic structure and molecular mechanisms at the DNA, RNA, and protein levels of the organisms (Manzoni et al., 2018). The omic sciences provide a possible relationship between genomics and genome; transcriptomics and transcriptome; proteomics and proteome; metabolomics and metabolome. It also provides a brief and better understanding of the physiological processes, etiology of a disease, and its diagnosis. But advances in techniques and technologies are also facing few limitations at procedural, as well as data interpretation level. Human Genome Project revolutionized the genome sequencing of livestock species; discovery of new genes involved disease development and its prevention (Hood and Rowen, 2013). Early attempts to construct whole-genome maps of livestock species were based on the two technologies, i.e., somatic cell genetics and in situ hybridization (Womack, 2005). These strategies were extremely important in early comparative genome mapping because the mapped markers were conserved across the mammalian genome. In the late 20th Century, genomics has opened a new opportunity with the sequencing of the human genome and showed that the complexity of life had been limited to the sequence of the nucleotides in DNA. At that time, bacterial restriction endonucleases were used to visualize the differences in the sequence of DNA and genome mapping. In 1985, with the development of polymerase chain reaction (PCR), anentirely new area opened to detect and study the differences in the sequence of various genes. In the early 1990s, PCR with genetic markers became a powerful tool to construct the genetic maps of the livestock genomes. With the beginning of the 21st Century, the human genome project was moving toward an initial draft of the human genome sequence, and additional technologies became available that allowed researchers to move into large-scale gene expression studies. The human genome project was completed in 2003, with a cost of approximately $18 million. The key findings of the human genome project were that the human genome contains about 3.2 billion nucleotide bases, approximately 20,000e25,000 genes, and in which chromosome 1 contains most genes,

* Present Address: Nilamber-Pitamber University, Medininagar, Palamu, Jharkhand, India.

Advances in Animal Genomics. https://doi.org/10.1016/B978-0-12-820595-2.00001-1 Copyright © 2021 Elsevier Inc. All rights reserved.

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FIGURE 1.1 The central dogma of omics science, which integrates information from structural genomics to transcriptomics, proteomics, and phenomics.

i.e., 3168, while chromosome Y contains fewest near about 344 genes. The agricultural research community was then able to capitalize on the infrastructure built by the human genome project to sequence the chicken (Gallus domesticus) and the bovine genome (Bos taurus) (Gibbs et al., 2002; Zimin et al., 2009). In the year 2006, draft genome sequences of chickens and cattle were completed, and a new milestone was created in the history of agricultural animal research (Gay et al., 2007).

1.2 Branches of animal genomics 1.2.1 Structural genomics Structural genomics is the study of three- dimensional structure of every protein encoded by genes. It includes the genetic and physical mapping and sequencing of the whole genome. The main aim of structural genomics is to solve the experimental structures of all possible protein folds (Skolnick et al., 2000).

1.2.2 Functional genomics Functional genomics deals with the structure, function, and regulation of all genes rather than the single gene of the genome and dynamic aspects such as gene transcription, translation, and proteineprotein interactions (Bunnik and Roch, 2013). The aim of functional genomics is to relate the complex relationship between genotype and phenotype at the genome level. Functional genomics gives an idea to understand the time and place where genes will express in different subtypes of cells, level of gene expression, gene expression regulation, and interaction of genes and its product, changes in gene expression during the onset of various diseases, and functional roles of different genes in cellular processes (Fig. 1.2).

1.2.3 Epigenomics Epigenomics is the study of reversible epigenetic modifications in a cell’s DNA or histone protein that affect gene expression without altering the DNA sequence (Wang and Chang, 2018). These modifications are termed as epigenetic because modifications were taking place on the DNA, i.e., epi “on top of” the genetic material “DNA.” Two of the most

FIGURE 1.2 Schematic overview of network analysis of the genome to understand the integration of information of transcriptome, proteome, metabolome, and how these interactions determine biological functions.

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characterized epigenetic modifications are DNA methylation and histone protein modification (Rivera and Ren, 2013). Epigenomic modifications play an important role in numerous cellular processes, such as in differentiation, growth, and development of various metabolic disorders (Laura, 2008) (Fig. 1.3).

1.2.4 Metagenomics Metagenomics is the study of genomic content that recovered directly from environmental samples. There are two basic types of Metagenomics studies. I. Metagenomics based on sequencing and analysis of DNA from environmental samples II. Metagenomics based on screening of particular function or activity The study of Metagenomic explored many novel microbial genes that are involved in the metabolism like energy acquisition, carbon, and nitrogen metabolism in natural environments that were not mentioned scientifically in previous literature. Metagenomics, based on sequencing, is applied to explore the structure of genome, identify the novel genes, and compare the organism genomes of different communities to establish the degree of diversity (Handelsman, 2004). Metagenomics, based on the functions, is a powerful experimental approach to identify the genes for their unique function (Thomas et al., 2012). Functional metagenomics starts with the construction and screening of metagenomic libraries. Cosmid or fosmid based libraries are created due to large size DNA carrying capacity and high cloning efficiency. Using the expression system allows the discovery of novel protein/enzymes whose functions could not be predicted by DNA sequence alone (Fig. 1.4).

1.2.5 Pharmacogenomics Pharmacogenomics covers the study of drug response patterns of a patient in the human population and their correlation with the other patient data. As its name reflects, it is the combination of the study of two branches of science, i.e., pharmacology and genomics. Drug response analysis in patients is carried out by analyzing the inherited genetic variation (Johnson, 2003). The inherited genetic variation can be observed by correlating gene expression or single-nucleotide

FIGURE 1.3 Role of epigenetic modification in the development of impaired physiological functions.

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FIGURE 1.4 Steps involved in the metagenomics for the analysis of metagenome.

polymorphisms of the population. Comparison of genome analysis drug response data allows patients to be clustered into drug response groups for the administration of appropriate drugs and dose to maximize efficacy with minimal adverse drug reactions (Fig. 1.5). The area of pharmacogenomic testing is rapidly growing, but it has some barriers to implement the pharmacogenomic testing into clinical practice due to legal, logistical, and knowledge-based problems. But now, genetic counselors are providing the information to the clinicians to implement testing data in their practice.

1.3 Genetic markers used in animal genomics Genetic markers are a specific DNA sequence with a known position on a chromosome that can be used to identify individuals or species. Eukaryotic genomes have some level of polymorphisms in DNA sequences between species and within a species. Three types of DNA polymorphisms i.e., restriction fragment length polymorphisms (RFLPs), microsatellites, and single nucleotide polymorphisms (SNPs), have been particularly categorized.

1.3.1 Restriction fragment length polymorphism (RFLP) RFLP is a technique in which organisms may be differentiated by the analysis of fragmented patterns of DNA fragments derived from cleavage with restriction endonucleases. In two organisms, different lengths of fragments are produced when the DNA is digested with a restriction enzyme due to the difference in the distance between the sites of cleavage of particular restriction endonucleases. In order to analyze the RFLP of two individuals, it is very necessary to determine the size of the fragmented DNA by gel electrophoresis and, after transfer to a membrane by Southern blotting. Radioactively labeled probes are used to identify the interest of fragments, and different lengths of restriction fragments are produced from the genome of different individuals. Nowadays, polymerase chain reaction (PCR) is more commonly used to compare to southern hybridization. In PCR, primers are designed to anneal either side of the polymorphic site using a probe that spans the polymorphic restriction site. Amplified fragment with the restriction enzyme then run in an agarose gel to analyze

FIGURE 1.5 Factors and role of genetic polymorphism in the determination of pharmacogenomics properties in humans.

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the pattern of DNA fragment. Analysis of the RFLP pattern revealed that there are insertions or deletions within the fragments, unequal crossing over, point mutations within the restriction enzyme recognition site, and DNA rearrangements during the evolutionary processes. RFLPs are widely used in the study of diversity and phylogenetic of closely related species, and the construction of genetic maps (Table 1.1).

1.3.2 Random amplified polymorphic DNA (RAPD) RAPD is a PCR based technology in which DNA polymorphism assay is based on the amplification of random DNA segments with single primers of the arbitrary nucleotide sequence. A single type of primer is used to anneals to the genomic DNA at two different sites on complementary strands. RAPD polymorphism is detected by using short synthetic oligonucleotides (10 bases long) of random sequences as primers in a PCR reaction. In a strain that has DNA sequences in its genomic, complementary to the primer oligonucleotides, PCR products will be detected in the gel by ethidium bromide staining, while in those strains that do not have the complementary sequences, no product will be detected. The application of RAPDs ranges from studies at the individual level, as well as closely related species and gene mapping studies also.

1.3.3 Microsatellites The term microsatellites was coined by Litt and Lutty (1989) and is also known as short tandem repeats or simple sequence repeats (STRs/SSRs). Microsatellites are clusters of shorter, usually less than 13 bp and 10 to 20 times repeated units. These types of sequences are also called VNTRs (Variable Number Tandem Repeats) due to variation in the number of repeating units at a particular locus. STRs are tandem repeats and are frequently distributed in all eukaryotic genomes. They show a large and stable polymorphism due to variation in the number of repeat units and are almost ideal molecular markers for genome mapping. RFLP and RAPD markers show limited variation between parents, especially in naturally inbreeding species. This limits the numbers of these markers that can be effectively mapped in a single cross. In contrast, microsatellite and minisatellite are hypervariable. Microsatellites are very informative markers and show a high level of polymorphism that can be used in population genetics studies, ranging from the individual level to that of closely related species and gene mapping studies.

1.3.4 Single nucleotide polymorphism (SNP) Single nucleotide polymorphism (SNP) is the change in the DNA sequence at single nucleotide (A, T, G, or C) among in the genome of members of a species. These are unique sequences in a genome wherein some individuals will have one nucleotide, and others have a different nucleotide. SNPs are very stable because of low mutation rates and can be used as important genetic markers. But SNPs have less informational content as compared to highly polymorphic microsatellite.

1.4 Techniques used in creating transgenic animals A transgenic animal is an animal that carries transgene or deliberate modification in its genome. To create the transgenic animal, constructed transgene need to be introduced into the animal’s genome with the help of recombinant DNA technology. Constructed transgene must be integrated to the host genome and should be stable so that they pass on to subsequent generations. Heritability changes are done by modification in the genome of its germline, and hence, they will carry the changes in all their somatic and germline cells. All offspring derived from this animal will have the transgene and completely transgenic. Genetic modifications are created in animals to solve a variety of purposes such as to gain knowledge about gene function and sequence of the genetic code, improve animal production traits, produce new animal products, study gene control in complex organisms and build genetic disease models. The techniques that are currently applied to produce transgenic animals are explained below.

1.4.1 Microinjection Currently, the microinjection of DNA is the preferred method for producing transgenic animals (Gordon et al., 1980). This technique is based on the injection of a foreign DNA into a fertilized oocyte. The constructed transgene integrates randomly into the host oocyte genome, and subsequently, the zygote continues with the embryonic development, and then the embryo is transferred to a foster mother and eventually develops a transgenic animal. Although this is not a 100% efficient procedure, a large number of microinjected fertilized eggs must be used. However, this method has few

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TABLE 1.1 Advantages and disadvantages of RFLP, RAPD and SSR as genetic markers in the analysis of the genome. Type of markers

Advantages

Disadvantages

Restriction fragment length polymorphism (RFLP)

-

Large range genome coverage Abundantly present in the genome Can be used across species Need no sequence information reproducibility high

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Need radioactive labeling for detection Need good quality of DNA in a large amount Difficult to automate More laborious as compared to RAPD

Random amplified polymorphic DNA (RAPD)

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Large range genome coverage Abundantly present in the genome Need no sequence information Easy automation Required less amount of DNA No radioactive labeling for detection Less time consuming

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Can not be used across species reproducibility low Dominant markers No information of probe or primer

Simple sequence repeat (SSR)

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Abundantly present in the genome Reproducibility high Medium range genome coverage Easy automation No radioactive labeling for detection

- Not well-tested - Can not be used across species - required sequence information

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limitations. The injected DNA integrates at random sites within the genome and multiple copies of the injected DNA are incorporated at one site. In some individuals, the transgene may not be expressed due to an unusual site of integration, which will disrupt the normal physiology of the animals. For identifying the transgenic animals, DNA from a small piece of the tail can be assayed by either southern blot hybridization or the polymerase chain reaction for the presence of the transgene (Smith and Murphy, 1993; Haruyama et al., 2009).

1.4.2 Somatic cell nuclear transfer (SCNT) The method of somatic cell nuclear transfer (SCNT), is also known as somatic cell cloning and is a popular technique for the production of transgenic animals. In the somatic cell nuclear transfer technique, the nucleus of a somatic (body) cell is transferred to the cytoplasm of an enucleated egg (an egg in which the nucleus has been removed). In an egg, the somatic nucleus is reprogrammed by egg cytoplasmic factors to become a zygote (fertilized egg) nucleus. The egg is allowed to develop up to the blastocyst stage, and at this point, a culture of embryonic stem cells (ESCs) can be created from the inner cell mass of the blastocyst. ESCs of humans, monkeys, and mice have been made by using SCNT. ESCs have potential applications in both medicine and research. This method can also be used to produce transgenic animals, with the additional benefit of targeted genetic manipulation.

1.4.3 Artificial chromosome transfer Artificial chromosomes are artificially created chromosomes having the properties of centromeres, telomeres, and origins of replication, and specified sequences required for their stable maintenance within the cell as autonomous, self-replicating chromosomes. Due to these properties, there is no need for integration of the transgene into the host genome (Kazuki and Oshimura, 2011). This technique is used to transfer very large size, complex genes or many small genes and regulatory elements to a target animal. The strategy of transfer of artificial chromosomes into the host cell and subsequent cloning of animals is similar to the SCNT approach. This technique has been proven by the transfer of human antibody genes of 10 mb in size with human artificial chromosome to the cattle, and the transferred chromosome was stable, and the antibody genes expressed to a certain extent in the transgenic animal.

1.4.4 Embryonic stem (ES) cell-based cloning and transgenesis Cells from blastocyst stage of a developing embryo can proliferate in cell culture and still retain the capability of differentiating into all other cell types, including germline cells, after they are reintroduced into another blastocyst embryo. Such cells are called pluripotent embryonic stem cells. In culture, ES cells can be easily engineered genetically without a change in their pluripotency. With this system, a functional transgene can be integrated at a specific site in the genome of ES cells. Transformed cells can be selected, grown, and used to generate transgenic animals. The insertion of a transgene at a specific, predetermined DNA site of the host genome is called gene targeting (Bouabe and Okkenhaug, 2013). The insertion of a transgene at a specific site of the genome is much more complex compared to the insertion of the transgene at random sites. However, gene targeting is a powerful and widely used technique to insert transgene into a specific site (knock-in) or inactivate specific genes (knock-out) or replace the endogenous version of a gene with a modified version.

1.4.5 Viral vector-mediated DNA transfer Transgenesis can also be done by retrovirus-derived vectors of lentiviruses class. In this method, genes that are essential for viral replication are deleted from the viral genome and retain only the capacity for integration of the viral genome into the host genome. The deleted viral genome part is occupied by the transgene of interest. Viruses carrying the modified vector are produced in vitro and subsequently injected into the perivitelline space of the zygote (or an unfertilized oocyte), resulting in the integration of the viral genome into the host genome. Over the other gene transfer methods, the use of retroviral vectors has an advantage in term of effective means of integrating the transgene into the genome of the recipient cell. However, these vectors transfer only small pieces (Up to 8 kb) of DNA. Due to size constraints, it may lack essential adjacent sequences for regulating the expression of the transgene.

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1.5 Application of animal genomics The applications of animal genomics are to generate transgenic animals with enhanced resistance to diseases. Animal genomics offers a variety of other techniques that contribute to improved animal health. The application of animal genomics includes the production of vaccines to immunize animals against diseases, providing nutritious food for a growing human population, improving the sustainability of animal agriculture and animal welfare, increasing animal fitness, and developing improved disease diagnostic tools (Fig. 1.6).

1.5.1 Livestock breeding industry Animal breeding, nowadays, is an important biotechnological tool that influenced the whole range of applications and genomic developments. The main goal of animal genomics is genetic progress within a population to improve the genetic resources, and ultimately, the phenotypic outcome (Dekkers, 2012). Genetic progress is influenced by several factors that include the age of breeding (generation interval), the proportion of the population selected for further breeding (selection intensity), the additive genetic variation within the population, and the accuracy of choosing candidates for breeding. In the above-mentioned factors, the first factor needs to be decreased, whereas the last three factors need to be increased in order to increase genetic progress. All the techniques that are helpful in genetic progress can be divided into two groups. All techniques that interfere with reproduction efficiency are kept in the first group, which includes multiple ovulation, artificial insemination, embryo sexing, ova pick-up, embryo transfer (ET), and cloning. With the use to these technologies, we can increase breeding accuracy, selection intensity, and shortened generation interval. The second group of techniques includes molecular determination of genetic variability and the identification of genetically valuable traits and characteristics (Tan et al., 2017). It has been suggested that selective breeding may be able to improve characters like resistance to disease and stresses, phenotypic appearance, and esthetic sensitivity. Production of transgenic livestock has significantly improved human health; enhanced nutrition with decreased livestock diseases.

1.5.2 Transgenic animal A transgenic animal is an animal that has a foreign gene in its genome by genetic engineering techniques. In the production of the transgenic animal, it is necessary to transfer the constructed recombinant DNA with the help of recombinant DNA technology. The transgene should be stable, i.e., attached to the chromosomal DNA, expressed and passed on to the next generations. Genetic changes are carried out in the germ cell, so they will carry this genetic modification in all their germ line cells, as well as all somatic cells (Kumar, 1994). This will result in stable transformation, and all offspring from the transformed animal will be completely transgenic animals.

FIGURE 1.6 Application of animal genomics.

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Transgenic animals have several application includes medical importance such as xerotransplantation (Heiner and Wilfried, 2003), animal model for in vivo study (Bagle et al., 2012), to gain knowledge of gene function and further decipher the genetic code, bioreactor for pharmaceutical, in the agricultural field, such as disease resistant animals, increased quantity, produce new animal products, and quality of the milk, meat, egg, and wool production, and to test the toxicity of chemicals against sensitive animals. Ralph Brinster (University of Pennsylvania) and Richard Palmiter (University of Washington) created the first transgenic animal “Supermouse” in 1982 by inserting the human growth hormone gene into the mouse fertilized, single-cell stage oocyte genome by the microinjection technique. Transgenic mice were unnaturally large as compared to parents, and insertion of a single growth hormone gene was sufficient to have tremendous effects on mice. Later on, several other animals such as pig, goat, cow, sheep, fish, etc. are developed transgenetically. It is very difficult to create a specific character in comparison to the simple transfer of responsible gene into the animal genome due to high costs, technological limitations, insufficient knowledge about gene function and regulation of gene expression. One major application of transgenic animals is the production of pharmaceutical products. Many human proteins cannot be produced in microorganisms because they lack posttranslational modification mechanisms that are responsible for the proper functioning of synthesized protein, low yield, and requirement of high man power. Transgenic animal bioreactors are an attractive alternative approach to produce the biopharmaceutical products such as body fluids, including urine, saliva, milk, blood, chicken egg white, etc.

1.5.3 Gene therapy Gene therapy may be defined as introduction of a functional gene into cells that contain the defective gene. The main aim of the human genome project was to understand the causative genes of all inherited diseases, the mechanism of etiology of diseases, and find out its therapy (Kaplan, 2002; Soutullo, 2004). The treatment of monogenic diseases can be treated easily, but the major challenge today is to understand and treat the polygenic and multifactorial etiology of common diseases, such as cardiovascular, cancer, nutritional, auto-immune, allergic, degenerative disorders. In most of the cases, when a gene gets mutated, it causes the onset of a specific disease. About 8000 different monogenic diseases have been listed in the McKusick catalog. Monogenic diseases are less complex and are called “Mendelian” diseases, whereas polygenic diseases are more complex and are called non-Mendelian diseases because segregation of the polygenic trait does not follow strictly Mendelian rules. More advances in genomics will help to understand the molecular mechanisms of all types of infections such as virulence, susceptibility/resistance to microbes, resistance to antibiotics, degenerative disorders, malignancies, neuropsychiatric illnesses, developmental diseases, etc. Application of gene therapy involves the following steps: 1. 2. 3. 4.

Identification of the gene that plays a key role in the development of a genetic disorder Determination of the role of its products in disease development Isolation and cloning of the gene Methods of gene therapy

The insertion of genetic material into a human being for the sole purpose of correcting a genetic defect, i.e., somatic cell gene therapy, is socially acceptable. The modification of germ cells by gene manipulation under in vitro is possible, and this process is considered as transgenesis. Gene therapy is used to correct the inborn error of metabolism by the insertion of a normal gene into the organism with the defective gene. But there are some criteria to select the genetic disorder for gene therapy. 1. 2. 3. 4.

Disease should be life threatening The gene responsible for the disease has been cloned A precise regulation of the gene should not be required Suitable gene delivery system should be available

1.5.4 Superovulation Superovulation is a reproductive technology used in the dairy industry to increase the reproductive rate of superior females. It is also called superstimulation. Superovulation is the primary requirement for physiologically low ovulation rates (cattle, sheep, goats, and horses) in animals for the successful application of embryo transfer. Superovulation is achieved by using follicle-stimulating gonadotropins, FSH and LH hormones to promote the development of subordinate follicles

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(Stouffer and Zelinski-Wooten, 2004). Superovulation with gonadotropins is an essential assisted reproductive technology that increases the number of oocytes to achieve high pregnancy rates. Oral or injectable administration of gonadotropins to females is widely used for treatment and increasing the number of offspring from animals. But numerous studies have strongly indicated that superovulation can lead to unhealthy oocyte maturation, impaired embryo development, decreased implantation rate, and increased postimplantation loss (Wurth et al., 1994; Rizos et al., 2002).

1.5.5 Improving hair and fiber With the help of transgenic manipulation, we are continually focusing on increasing the quality, yield, length, strength, fineness, the color of hair, wool, and fiber for fabric and yarn production. Recently, a novel approach to producing useful spider silk has been achieved using the milk of transgenic goats (Karatzas et al., 1999). Spiders produces seven different types of silk for making the webs in which the most durable variety is the dragline silk. This material has tensile properties close to synthetic fiber Kevlar and can be elongated up to 35% in length. There are many important applications of these fibers in medical devices, ballistic protection, sutures, aircraft, automotive composites, airbags, and in clothing.

1.5.6 Disease resistant animals Disease resistance is a condition in which an animal remains healthy even under the exposure of pathogenic agents. The availability of alternatives such as vaccination also increases the resistance against the disease. Resistance or susceptibility to diseases of an animal depends on the presence of a variety of the genes, but the identification of specific gene-related immune systems is an important aspect for reducing the occurrence of diseases. To reduce the onset of diseases in animals, scientists are now working either by introducing resistance genes or removing susceptibility genes from the animal genome. In transgenic animals, specific tissue or cells of an animal do not express the receptor that allows the pathogen to bind to cells, which is the primary step to cause any infection. Resistance against mastitis is a popular example of reducing the onset of diseases. Mastitis is a bacterial infection of the bovine mammary gland, leading to decreased productivity and milk contamination. A gene that is responsible for the synthesis of lysostaphin, a protein, is transferred to the cattle genome that is a potent inhibitor of Staphylococcus aureus, responsible for the majority of mastitis cases. According to available reports, the first transgenic cows have been produced, which are resistant to S. aureus mediated mastitis and secrete lysostaphin, small proteins, in their milk to reduce the bacterial infections (Donovan et al., 2006).

1.5.7 Nutritious food Human health of a population is directly affected by sustainable and secure supply of healthy food. For several years, scientists and farmers are working to improve livestock in terms of quantity, nutritious, and cost-effective animal products. Transgenesis are carried out to improve the nutrients in animal products, i.e., quality and quantity of the animal products and specific nutritional composition. Genetic modification via enhanced nutrition, transgenic animal products will play significant roles in improving public health. Omega-3 fatty acid, a fish product, is used to decrease the occurrence of coronary heart disease. Now, with the help of animal genomics, transgenic pigs have been producing elevated levels of omega-3 fatty acids (Lai et al., 2006). Transfer of a gene responsible for elevated levels of omega-3 fatty acids into pigs may enhance the nutritional quality of pork also. Advances in transgenic technology provide the opportunity to improve the composition of milk with a greater quantity of milk, higher nutrient content, and milk that contains a beneficial “nutriceutical” protein, as well as defensive protein such as lactoferrin (Hyvonen et al., 2006; Wheeler, 2013). This application of transgenic technology will also help the growth and survival of offspring.

1.6 Conclusion Transgenic animals that carry genetically engineered genes from other species have great potential to improve human welfare. DNA microinjection, embryonic stem cell-mediated gene transfer, retrovirus-mediated gene transfer, and artificial chromosome transfer are some popular methods used to produce transgenic animals. Transgenic technology holds great potential in many fields, mainly in agriculture, medicine, and industry. Human welfare and ethical concerns will determine the acceptability of genome editing to consumers, and genetic manipulation that will benefits the animals will be more acceptable to the public. Applications that benefit the animals are more acceptable to the public. The use of genome editing to produce transgenic animal with a direct welfare impact has improved animal health, well-being, production efficiency, and product quality in ways that meet the demands of the growing global populations.

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References Bagle, T.R., Kunkulol, R.R., Baig, M.S., More, S.Y., 2012. Transgenic animals and their application in medicine. Int. J. Med. Res. Health Sci. 2 (1), 107e116. Bouabe, H., Okkenhaug, K., 2013. Gene targeting in mice: a review. Methods Mol. Biol. 1064, 315e336. Bunnik, E.M., Roch, K.G.L., 2013. An introduction to functional genomics and systems biology. Adv. Wound Care (New Rochelle) 2 (9), 490e498. Dekkers, J.C.M., 2012. Application of genomics tools to animal breeding. Curr. Genom. 13, 207e212. Donovan, M.D., Lardeo, M., Foster-Frey, J., 2006. Lysis of staphylococcal mastitis pathogens by bacteriophage phi11 endolysin. FEMS Microbiol. Lett. 265 (1), 133e139. Gay, C.G., Zuerner, R., Bannantine, J.P., Lillehoj, H.S., Zhu, J.J., Green, R., Pastoret, P.P., 2007. Genomics and vaccine development. Rev. Sci. Tech. Off. Int. Epiz. 26 (1), 49e67. Gibbs, R.A., Weinstock, G., Kappes, S.M., Schook, L.B., Skow, L., Womack, J., 2002. Bovine Genomic Sequencing Initiative: De-humanizing the Cattle Genome. Available at: http://www.genome.gov/Pages/Research/Sequencing/SeqPr oposals/BovSeq.pdf. Gordon, J.W., Scangos, G.A., Plotkin, D.J., Barbosa, J.A., Ruddle, F.H., 1980. Genetic transformation of mouse embryos by microinjection of purified DNA. Proc. Natl. Acad. Sci. U.S.A. 77 (12), 7380e7384. Handelsman, J., 2004. Metagenomics: application of genomics to uncultured microorganisms. Microbiol. Mol. Biol. R. 68 (4), 669e685. Haruyama, N., Cho, A., Kulkarni, A.B., 2009. Overview: engineering transgenic constructs and mice. Curr. Protoc. Cell. Biol. CHAPTER: Unite19.10. Heiner, N., Wilfried, A.K., 2003. Application of transgenesis in livestock for agriculture and biomedicine. Anim. Reprod. Sci. 79, 291e317. Hood, L., Rowen, L., 2013. The Human genome project: big science transforms biology and medicine. Genome Med. 5 (9), 79. Hyvonen, P., Suojala, L., Orro, T., Haaranen, J., Simola, O., Rontved, C., Pyoroala, S., 2006. Transgenic cows that produce recombinant human lactoferrin in milk are not protected from experimental Escherichia coli intramammary infection. Infect. Immun. 74 (11), 6206e6212. Johnson, J.A., 2003. Pharmacogenetics: potential for individualized drug therapy through genetics. Trends Genet. 19 (11), 660e666. Kaplan, J., 2002. Genomics and medicine: hopes and challenges. Gene Ther. 9, 658e661. Karatzas, C.N., Zhou, J.F., Huang, Y., Duguay, F., Chretien, N., Bhatia, B., Bilodeau, A., et al., 1999. Production of recombinant spider silk (BioSteelÒ) in the milk of transgenic animals. Transgenic Res. 8, 476e477. Kazuki, Y., Oshimura, M., 2011. Human artificial chromosomes for gene delivery and the development of animal models. Mol. Ther. 19 (9), 1591e1601. Kumar, R., 1994. Therapeutic applications of transgenic animals. In: Kobayashi, T., Kitagawa, Y., Okumura, K. (Eds.), Animal Cell Technology: Basic & Applied Aspects. The Sixth International Meeting of Japanese Association for Animal Cell Technology JAACT’93, vol. 6. Springer, Dordrecht. Lai, L., Kang, J.X., Li, R., Wang, J., Witt, W.T., Yong, H.Y., Hao, Y., Wax, D.M., Murphy, C.N., Rieke, A., Samuel, M., Linville, M.L., Korte, S.W., Evans, R.W., Starzl, T.E., Prather, R.S., Dai, Y., 2006. Generation of cloned transgenic pigs rich in omega-3 fatty acids. Nat. Biotechnol. 24 (4), 435e436. Laura, B., 2008. Epigenomics: the new tool in studying complex diseases. Nat. Edu. 1 (1), 178. Laura, B., 1989. A hyper variable microsatellite revealed by in vitro amplification of dinucleotide repeats within cardiac muscle actin gene. Am. J. Hum. Genet. 44, 397e401. Manzoni, C., Kia, D.A., Vandrovcova, J., Hardy, J., Wood, N.W., Lewis, P.A., Ferrari, R., 2018. Genome, transcriptome and proteome: the rise of omics data and their integration in biomedical sciences. Brief. Bioinform. 19 (2), 286e302. Rivera, C.M., Ren, B., 2013. Mapping human epigenomes. Cell 155, 39e55. Rizos, D., Ward, F., Duffy, P., Boland, M.P., Lonergan, P., 2002. Consequences of bovine oocyte maturation, fertilization or early embryo development in vitro versus in vivo: implications for blastocyst yield and blastocyst quality. Mol. Reprod. Dev. 61 (2), 234e248. Skolnick, J., Fetrow, J., Kolinski, A., 2000. Structural genomics and its importance for gene function analysis. Nat. Biotechnol. 18, 283e287. Smith, D.R., Murphy, D., 1993. Genomic analysis of transgenic animals: southern blotting. Methods Mol. Biol. 18, 323e327. Soutullo, D., 2004. Gene therapy, yesterday and nowadays. In: Sussane, C. (Ed.), Societal Responsibilities in Life Sciences, vol. 12. Kamla Raj Enterprises, New Delhi, India, pp. 59e67. Stouffer, R.L., Zelinski-Wooten, M.B., Meyer, F., 2004. Overriding follicle selection in controlled ovarian stimulation protocols: quality vs quantity. Reprod. Biol. Endocrinol. 2, 32. Tan, C., Bian, C., Yang, D., Li, N., Wu, Z.F., Hu, X.X., 2017. Application of genomic selection in farm animal breeding. Yi Chuan 39 (11), 1033e1045. Thomas, T., Gilbert, J., Meyer, F., 2012. Metagenomics - a guide from sampling to data analysis. Microb. Inf. Exp. 2, 3. Wang, K.C., Chang, H.Y., 2018. Epigenomicsdtechnologies and applications. Circ. Res. 122 (9), 1191e1199. Wheeler, M.B., 2013. Transgenic animals in agriculture. Nat. Edu. Knowl. 4 (11), 1. Womack, J.E., 2005. Advances in livestock genomics: opening the barn door. Genome Res. 15 (12), 1699e1705. Wurth, Y.,A., Merton, S., Kruip, T., 1994. In Vitro Maturation and Fertilization Limit the Embryo Production Rate and in Vitro Embryo Development Diminishes Embryo Viability. Utrecht, The Netherlands (Thesis). Zimin, A.V., Delcher, A.L., Florea, L., Kelley, D.R., Schatz, M.C., Puiu, D., Hanrahan, F., Pertea, G., Tassell, C.P.V., Sonstegard, T.S., Marçais, G., Roberts, M., Subramanian, P., Yorke, J.A., Salzberg, S.L., 2009. A whole-genome assembly of the domestic cow, Bos Taurus. Genome Biol. 10, R42.

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Further reading Vidana, S., Rajwani, R., Wong, M.S., 2017. The use of omic technologies applied to traditional Chinese medicine research. Evid. Based. Complement. Alternat. Med. 6359730.

Chapter 2

From gene to genomics: tools for improvement of animals Pradeep Kumar Singh1, Pankaj Singh2, Rajat Pratap Singh3 and Ram Lakhan Singh1, 4 1

Department of Biochemistry, Dr. Rammanohar Lohia Avadh University, Ayodhya, Uttar Pradesh, India; 2Department of Biotechnology,

Dr. Rammanohar Lohia Avadh University, Ayodhya, Uttar Pradesh, India; 3Department of Biotechnology, Guru Ghasidas University, Bilaspur, Chhattisgarh, India; 4Vice-Chancellor, Nilamber-Pitamber University, Medininagar, Palamu, Jharkhand, India

2.1 Introduction All animals possess a linear double-stranded DNA molecule as the genetic material without exception. Small segments of DNA that are able to form primary transcript or functional proteins are known as genes. Genes are the source of phenotypic variation in all living organisms. Complete set of DNA, including its entire gene, represent the genome of an organism. Genomes vary in size. The smallest known genome of a bacterium contains about 600,000 DNA base pairs, while human genomes have some three billion DNA base pairs. All human cells except for mature erythrocytes, possess a complete genome. More than 65 years have been completed since the 1953 landmark description of the DNA double helix by Watson and Crick. The individual work of the Human Genome Project and Celera Genomics has successfully sequenced the human genome (Venter et al., 2001; Lander et al., 2001) and opened the door for the postgenomic era (Guttmacher and Collins, 2003). As we know that only 1.1% of the genome consists of exons coding for proteins, 24% is intronic sequences, and the remaining 75% consists of intergenic DNA. Thus more than 98% human genome is without a known function, and it is considered as intron champion. In comparison with evolutionary lower organisms, it was found that human beings have only two to three times as many genes as the fruit fly and the mustard plant. This indicates the functional complexity rather than the absolute number of genes is required for the human phenotype.

2.2 Genes The fundamental physical and functional unit of heredity is the gene. It consists of a specific sequence of nucleotides that code for a specific protein. The size of genes in higher eukaryotes varies greatly. Genes consist of three types of regions: l

l

l

Noncoding regions, called introns, which do not specify amino acids and are removed (spliced) from the mRNA molecule before translation (Fig. 2.1). Coding regions, called exons, which specify a sequence of amino acids and collectively determine the amino acid sequence of the protein product. These portions of the gene are represented in the final mature mRNA molecule. Regulatory sequences, which play an important role in regulation of gene expression

Genes are made up of deoxyribonucleic acid (DNA) and act as instructors to make molecules called proteins. Genes vary in size due to numbers of nucleotides that vary from gene to gene, which may be a few hundred DNA bases to more than two million bases. Most of the portion of a gene in higher eukaryotes consists of noncoding DNA that interrupts the relatively short segment of the coding DNA. The Human Genome Project estimated that humans have nearly 20,000 to 25,000 genes located on 46 chromosomes (23 pairs) (Phillips, 2008; Finegold, 2017). These genes are collectively known as the human genome. The number of genes in an organism’s genome varies significantly between species. For example, the human genome contains an estimated 20,000 to 25,000 genes, whereas the genome of the bacterium Escherichia coli contains 5416 genes. In eukaryotes (animals, plants, and fungi), genes are mainly located within the cell nucleus, but

Advances in Animal Genomics. https://doi.org/10.1016/B978-0-12-820595-2.00002-3 Copyright © 2021 Elsevier Inc. All rights reserved.

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FIGURE 2.1 A structural gene involves a number of different components.

cell organelles mitochondria (animals and plants) and the chloroplasts (in plants) also contain small subsets of genes in addition to the genes found in the nucleus. In prokaryotes that lack a well-developed nucleus, genes are present in the cell cytoplasm on a single chromosome. Many bacteria also contain extrachromosomal genetic elements (plasmid) with a small number of genes.

2.2.1 Chromosome structure and organization Eukaryotic DNA is tightly packed into structures called chromosomes, consisting of long chains of DNA and histone proteins. Histone proteins provide structural support and play a role in controlling the activities of the genes. On average, each human chromosome’s DNA strand is about 4.3 cm long (Schaefer and Thompson, 2014). In one strand, 150 to 200 nucleotides long chain is wrapped twice around a core of eight histone proteins to form a structure called a nucleosome. The nucleosome is the histone octamer structure at the center and made up of two units each H2A, H2B, H3, and H4 histone proteins. The chains of histones proteins are again coiled to form a solenoid structure, which is stabilized by the histone H1protein. Further supercoiling of the solenoid structure formed more condensed structure chromosome. During cell cycle, each chromosome has two chromatids. Chromosomes and the DNA they contain are duplicated and passed to the daughter cells through the processes of mitosis and meiosis. Human beings have 22 pairs of autosomes and a pair of sex chromosomes, two X sex chromosomes for females (XX) and an X and Y sex chromosome for males (XY). The process of pairing and ordering all the chromosome of an organism is known as karyotyping. The chromosomes are arranged in decreasing size order from chromosome number 1 to 22 (Fig. 2.2). The prokaryotic cells lack a discrete nucleus; hence, chromosomes of prokaryotic cells are not enclosed by a separate membrane. Mostly, bacteria have a single, circular chromosome but exceptionally Streptomyces has linear chromosome, whereas Vibrio cholera has two circular chromosomes. Chromosome together with ribosomes and proteins located in a region of the cell cytoplasm is called nucleoid. Prokaryotic genomes are more compact as compared to eukaryotes. Prokaryotic genomes lack introns, and the genes tend to be expressed in groups known as operons.

Strands

Nucleus

5’ 3’ Mitochondria Cell 1

6

13 19

2

7

Base pair 3

8

14 15 20

4

9

5

Sugar phosphate backbone

10 11 12

16 21 22

17 18

23

3’ 5’

DNA Double helix

XY

Pairs of chromosomes ina human cell

FIGURE 2.2 Chromosomes in human Cell and DNA structure.

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2.2.2 Gene structure and organization 2.2.2.1 Eukaryotic gene Genes are composed of deoxyribonucleic acid (DNA). A DNA molecule is composed of two chains of polynucleotides and form a helical structure. The backbone of two side chains of DNA is made up of sugars and phosphates and bonded by pairs of nitrogenous bases. Nitrogenous bases include adenine (A), guanine (G), cytosine (C), and thymine (T) in which adenine is specifically bonded to thymine with two hydrogen bond. Similarly, the cytosine of one chain binds to the guanine of the other chain with three hydrogen bonds. Gene, which contains the necessary information for survival, is the specific polynucleotide sequence (Alberts et al., 2002; Polyak and Meyerson, 2003). In most organisms, genes are made of DNA, where the specific DNA sequence determines the function of the gene. A gene is transcribed from DNA into RNA, which can either be noncoding RNA (example: rRNA and tRNA) with a direct function, or an intermediate messenger (mRNA) that is then translated into protein (Fig. 2.3). Each gene has multiple specific sequence elements, which control the information flow in the genetic system and functional activities of the gene (Polyak and Meyerson, 2003). The length of these regulatory sequences may be as short as a few base pairs or up to many thousands of base pairs long. Much of gene structure in eukaryotes and prokaryotes is broadly similar. The presence of common elements largely results due to sharing ancestry of cellular life in organisms over two billion years ago (Werner and Grohmann, 2011). Understanding gene structure is the basic requirement to understand the gene expression, function, and annotation (Alberts et al., 2002). Structural differences in gene structure of eukaryotes and prokaryotes directly indicate their divergent transcription and translation machinery from the original (Kozak, 1999; Struhl, 1999). Gene expression is more tightly regulated and typically has more regulatory elements in eukaryotic genes as compared to prokaryotes (Struhl, 1999). There are certain regulatory structures in eukaryotic genes, which are not found in prokaryotes. Post-transcriptional modification of pre-mRNAs produces mature mRNA, which is translated into protein. In multicellular eukaryotes, for example, humans, all the genes are not expressed in all cells but gene expression varies widely among different tissues (Maston et al., 2006). The key structural feature of eukaryotic genes is that their transcripts are typically subdivided into exon and intron regions. Exon regions are retained in the final mature mRNA molecule and

Regulatory sequences

Enhancer/ Silencer

Promoter Proximal Core

Regulatory sequences

Open reading frame

5’UTR

Stop

Start

Exon 1

3’UTR

Exon 2

Exon 3

Enhancer/ Silencer

Terminator

Transcription Pre-mRNA

Intron 1 5’cap

Intron 2

Protein coding region

mRNA

Post-transcriptional modification Poly A tail

Translation Protein

Protein FIGURE 2.3 Structure of the eukaryotic protein-coding gene. Regulatory sequence(Promoter and enhancer regions) controls the rate and location of gene expression (blue [light black in print version]). Post-transcriptional modifications modify the pre-mRNA to remove introns (white) and add a 50 cap and poly-A tail. Finally, mature mRNA translates into the protein product.

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instruct the translation process, while during post-transcriptional processing, intron regions are spliced out (Matera and Wang, 2014). In post-transcriptional processing, the exons form a single continuous protein-coding region, and the splice boundaries are not detectable. 50 capping to the start of the mRNA and a poly-adenosine tail to the end of the mRNA is the major post-transcriptional modification, which stabilizes the mRNA and guides its transport from the nucleus to the cytoplasm (Guhaniyogi and Brewer, 2001). In genes, there are multiple enhancer and silencer sequences to which an activator or repressor protein binds and modifies the expression of genes (Maston et al., 2006; Pennacchio et al., 2013). Enhancers and silencers may be thousands of base pairs long and distantly located from the gene. Regulatory elements can also overlap one another, where many competing activators and repressors, as well as RNA polymerase, may be able to interact with gene regulatory sequences. For example, some repressor proteins can bind to the core promoter to prevent polymerase binding (Ogbourne and Antalis, 1998). Binding of activators and repressors to multiple regulatory sequences have a cooperative effect on transcription initiation (Kazemian et al., 2013). The binding of different transcription factors to regulator sequences influence the rate of transcription initiation at different times and in different cells (Maston et al., 2006). The core promoter marks the start site for transcription by binding RNA polymerase and other proteins necessary for copying DNA to RNA (Thomas and Chiang, 2008; Juven-Gershon et al., 2008).

2.2.2.2 Prokaryotic gene The arrangement of prokaryotic genes is entirely different from that of the eukaryotes in which genes are arranged into a polycistronic operon controlled by a set of regulatory sequences (Fig. 2.4). A group of genes that served related functions are transcribed onto the same mRNA, and hence, are coregulated (Jacob and Monod, 1961; Salgado et al., 2000). Typically, each gene in a polycistronic operon has its own ribosome binding site (RBS), resulting in simultaneous translation of all proteins on the same mRNA. Prokaryotic mRNA has multiple ORFs (open reading frames) in a single mRNA. In a polycistronic operon, the transcription and translation take place at the same time and in the same subcellular location (Salgado et al., 2000; Lewis, 2005). Some operons also display translational coupling, where the translation rates of multiple ORFs within an operon are linked (Levin-Karp et al., 2013; Tian and Salis, 2015). This can occur when the ribosome remains attached at the end of an ORF (Schumperli et al., 1982).

Polycistronic operon

Regulatory sequences

Regulatory sequences

Enhancet/ Silencer

Operator Promoter 5’UTR

Open reading frame UTR Start

Open reading frame Start

Stop

Stop

3’UTR

Enhancet/ Silencer

Terminator

Transcription RBS

Protein coding region

RBS

Protein coding region

Translation

Protein

Protein

FIGURE 2.4 Structure of a prokaryotic operon of protein-coding genes. Regulatory sequence (Promoter and enhancer regions) controls the rate of multiple protein-coding gene expression (blue [light black in print version]). The mRNA is translated into the final protein products.

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Transcription of the gene into an mRNA is regulated by the promoter, operator, and enhancer regions. The flanking untranslated regions (UTRs) of mRNA contain regulatory sequences responsible for the regulation of translation (Guhaniyogi and Brewer, 2001; Shafee and Lowe, 2017). Final protein products are encoded by the region between start and stop codons. The 3’ UTR contains a terminator sequence, which marks the endpoint for transcription and releases the RNA polymerase while 50 UTR binds the ribosome (Kuehner et al., 2011). In the case of genes for noncoding RNAs, the RNA is not translated but instead folded to be directly functional (Mattick, 2006; Palazzo and Lee, 2015).

2.3 Genome 2.3.1 Anatomy of the eukaryotic genome Genome is the complete set of hereditary material (DNA) of an organism. Every genome contains entire information expected to develop and keep up that life form. In eukaryotic cells, a large portion of DNA is present in the nucleus in the form of extensively folded structures known as chromosomes. Every chromosome is made up of a linear DNA molecule associated with specific proteins. Eukaryotic genomes are composed of one or more chromosomes. The number of chromosomes fluctuates broadly from species to species. In addition to the nuclear chromosomes, chloroplasts and mitochondria have their own DNA. In eukaryotic cells, the nuclear DNA-protein complex is organized in a compact way and packed as chromatin (Ridgway and Almouzni, 2001). The nucleosome is the basic unit of chromatin. A nucleosome comprises of DNA twisted around a protein octamer core. The octamer core consists of two of each of four highly evolutionary conserved core histones H2A, H2B, H3, and H4. The DNA between each histone octamer is known as linker DNA. The eukaryotic genome includes gene and gene-related sequences and intergenic sequences (Fig. 2.5).

2.3.1.1 Gene and gene-related sequences 2.3.1.1.1 Exons (protein-coding regions) These are the DNA sequences that carry the instructions to make proteins. The extent of the genome occupied by coding sequences varies extensively.

Eukaryotic Genome

Gene and Gene related sequences

Exons

Regulatory sequences Example: Promoter

Intergenic DNA

Gene related sequences

Genome wide repeats Examples: Transposan

Other Intergenic regions

Microsatellite

Introns

Gene Fragments

Pseudo Genes

Minisatellite

Unique Sequences FIGURE 2.5 Anatomy of the eukaryotic genome.

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2.3.1.1.2 Regulating sequences It includes promoters, enhancers, silencers, and other sequences, which regulate the transcription of genes. Promoters are short sequence elements, which facilitate the transcription initiation. Enhancers are certain positive transcriptional control elements, and silencers serve to diminish the transcription levels. 2.3.1.1.3 Introns These are noncoding DNA sequences within a gene, which are transcribed into the corresponding sequence in RNA transcripts. The introns are classified into four groups, Group I, Group II, Group III, and Group IV. The Groups I and II introns are self-splicing introns. 2.3.1.1.4 Gene fragments These are the pieces of genes containing only the exons. 2.3.1.1.5 Pseudogenes Pseudogenes are nonfunctional relatives of genes that have lost their ability to code protein. Pseudogenes arise from the collection of multiple mutations within a gene whose product is not required for the endurance of the life form.

2.3.2 Sequencing genomes Haemophilus influenzae was the first organism whose entire genome was sequenced in 1995 (Fleischmann et al., 1995). The first eukaryotic organism to be sequenced was Saccharomyces cerevisiae in 1996. Whole-genome sequencing is apparently the way toward determining the complete nucleotide sequence of an organism’s genome at a single time. It provides the raw DNA sequence of an individual organism’s genome. However, further analysis must be performed to give the biological meaning of this sequence. Genome sequencing provides significant information about the genes. It also helps scientists to understand the functioning of the genome to coordinate the development and maintenance of an entire organism. The entire genome sequence will help to study the functional genes and their interaction, regulatory regions, and junk DNA of the genome. A large number of sequencing experiments must be done so as to decide the sequence of an entire genome. There are two different approaches to sequence the eukaryotic genomes.

2.3.2.1 Shotgun approach It was developed by Fred Sanger in 1982. In this approach, the genome is randomly broken into small fragments, followed by the sequencing of each fragment. The resulting sequences are examined for overlaps. The fragments are reassembled on the basis of sequence overlaps into the full genome sequence (Fig. 2.6). The key necessity of the shotgun approach is that it must be conceivable to recognize overlaps between all the individual sequences that are produced. The identification procedure must be precise and straightforward to obtain the correct genome sequence. A mistake in identifying a pair of overlapping sequences could prompt the genome sequence to get mixed, or parts being missed out completely. The likelihood of committing errors increases with larger genome sizes, so the shotgun approach has been used predominantly with the smaller bacterial genomes.

2.3.2.2 Clone contig approach or clone by clone approach The clone contig approach is used for the sequencing of larger genomes. This approach includes a presequencing stage during which a progression of overlapping clones is recognized. A physical map of the entire genome is constructed with the help of restriction enzymes. Each fragment of genomic DNA is cloned utilizing a suitable host-vector framework. The cloning system used for building physical maps of large genomic regions is generally yeast artificial chromosomes (YACs) and bacterial artificial chromosomes (BACs) or the closely related P1-derived artificial chromosomes (PACs) (Burke et al., 1987; Green et al., 1998; Shizuya et al., 1992; Ioannou et al., 1994). The individual clones are then analyzed for the presence of unique DNA spots that are utilized to assemble overlapping clone maps. This contiguous series is called a contig. Every clone contig contains the DNA from a contiguous segment of the genome. Each piece of cloned DNA is then sequenced, and this sequence placed at its appropriate position on the contig map so as to continuously develop the overlapping genome sequence (Fig. 2.6). The downside of clone contig approach is that it includes a considerably more work, and thus, takes a long time and is more expensive.

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Genomic DNA

Shotgun Approach

Clone Contig Approach

Break genomic into large fragment by restriction enzyme

Break genomic into smaller fragment by sonication

Sequencing

Each fragment is cloned in a suitable host-vector system

Examined for overlaps

Organized mapped large clone contigs

Assembled on the basis of sequence overlaps

Sequencing of each clone

Assemble sequences of overlapping clones FIGURE 2.6 Genome sequencing approaches: Shotgun and Clone contig approach.

2.3.3 The methodology for DNA sequencing DNA sequencing is the way toward determining the arrangement of nucleotides in a fragment of DNA. The basic techniques of DNA sequencing are the chemical method (Maxam-Gilbert sequencing) and chain termination method or dideoxy method (Sanger’s method). More up to date techniques that can process a large number of DNA molecules rapidly are collectively called Next-Generation Sequencing (NGS) methods or High-Throughput Sequencing (HTS) techniques.

2.3.3.1 Chemical method (Maxam-Gilbert sequencing) Allan Maxam and Walter Gilbert developed a DNA sequencing method in 1976e77, based on chemical modification of DNA and subsequent cleavage at specific bases. It is a two-step catalytic process that is used for the sequencing of singlestranded DNA involving piperidine and two chemicals (dimethyl sulfate and hydrazine) that selectively attack purines and pyrimidines (Maxam and Gilbert, 1977). Purines react with dimethyl sulfate, and pyrimidines react with hydrazine in such a manner in order to break the glycoside bond between the ribose sugar and the base, displacing the base. Piperidine catalyzes the cleavage of the phosphodiester bond, where the base has been displaced. This technique requires radioactive labeling of a single-stranded DNA substrate at the 50 end. This marked substrate is exposed to four separate cleavage reactions (G, A þ G, C, C þ T) using specific chemicals, which generates a population of labeled cleavage products. The chemical treatment creates breaks at a small proportion of one or two of the four nucleotides dependent on each of four reactions. The fragments in the four reactions are loaded on high percentage polyacrylamide, and the fragments are resolved by gel electrophoresis. The fragments are visualized with the help of autoradiography that shows a series of bands, each relating to a radiolabeled DNA fragment, from which the sequence can be deduced. This technology has the disadvantage of relying on toxic chemicals.

2.3.3.2 Chain termination method or dideoxy method (Sanger’s method) Chain termination method is called Sanger’s method named after its pioneer Frederick Sanger who was awarded Nobel Prize in chemistry for this achievement in 1980. This method is also called the dideoxy method. Sanger’s method is based on the use of dideoxynucleotide triphosphates (ddNTPs) as DNA chain terminators, which lack the hydroxyl group at the 30 position. These modified nucleotides inhibit the addition of further nucleotides because they lack a 30 hydroxyl group, which involve in the formation of the phosphodiester bond with the incoming nucleotide. Thus the elongating DNA chain is terminated. The DNA sample to be sequenced is divided into four separate sequencing reactions. Each reaction contains a single-stranded DNA template, a DNA primer, a DNA polymerase, and standard deoxynucleotides. The primer or one of

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the nucleotides should be radioactively or fluorescently labeled to detect the final product. All of the four reactions contain a different ddNTP in much smaller amounts than the standard nucleotides. These ddNTPs terminate the DNA strand elongation. As the DNA is synthesized, occasionally, a dideoxynucleotide is integrating on to the growing chain by the DNA polymerase, which results in a chain-terminating event. Consequently, in a reaction mixture where the same chain of DNA is being synthesized over and over again, fragments of different lengths are produced due to the integration of the dideoxynucleotides at all possible positions (Russell, 2002). Once these reactions are completed, the newly synthesized and labeled DNA fragments are denatured and separated according to their length by gel electrophoresis using polyacrylamide gel. Each of the four reactions run in individual lanes. The gel is exposed to either UV light or X-ray to visualize the bands, depending on the method used for labeling the DNA. The relative positions of the different bands in all the four lanes are used to interpret the DNA sequence.

2.3.3.3 Next-generation sequencing (NGS) methods or high-throughput sequencing (HTS) Various new techniques for DNA sequencing were developed in recent years. These new DNA sequencing technologies are collectively considered as Next-Generation Sequencing (NGS) Technologies or High-Throughput Sequencing (HTS) Technologies. NGS is a powerful platform that has empowered the high-throughput sequencing of millions of small fragments of DNA from multiple samples in parallel. There are a variety of NGS technologies that use different strategies. In any case, most share a common set of features. Conceptually, NGS is somewhat similar to running many Sanger sequencing reactions in parallel. NGS can be used to sequence an entire or specific region of genomes. It is also capable of producing sequences with extremely high throughput and at a much lower cost than the classical sequencing technologies. The prominent NGS methods that are receiving adequate consideration are Illumina sequencing, Roche 454 Genome Sequencing, Pyrosequencing, Solid sequencing, Ion semiconductor sequencing, DNA nanoball sequencing, and few more (Porreca, 2010).

2.3.4 The Human Genome Projects The Human Genome Project (HGP) was an international scientific collaborative research project. It was designed to determine the DNA sequence and mapping of the genes of the entire human genome. The planning of HGP was started in 1984. It was finally launched in 1990 and completed on April 14, 2003 (IHGSC, 2004). A working draft of the human genome was reported in 2000, and a complete draft was published in 2003. According to the draft, approximately 22,500 protein-coding genes are present in the human genome (Pertea and Salzberg, 2010). The major funding agencies for HGP were the Department of Energy and National Human Genome Research Institute (NHGRI) at the National Institutes of Health (NIH) of the US government, as well as various other groups from around the world. The cost of this project was USD3-billion. It is the world’s largest collaborative biological project till date. Several other countries such as United Kingdom, Japan, France, Germany, Israel, and China, have contributed their scientific support and technological activities in the completion of this project (DeLisi, 2008). The genetic and physical mapping, followed by the sequencing of the human genome involves several essential steps. The genome was fragmented into smaller pieces of approx 150,000 base pairs in length. These fragments were ligated into BACs (bacterial artificial chromosomes) vector. The recombinant vectors were inserted into the bacteria, where they were amplified by the bacterial DNA replication machinery. Each of these pieces was sequenced by using the shotgun approach of genome sequencing.

2.3.5 Genomic libraries A genomic library is a set of clones that represent the entire genome of an organism. It contains all the genes and generelated sequences and intergenic DNA sequences. Construction of a genomic DNA library starts with isolation and purification of genomic DNA (Fig. 2.7). The genomic DNA is digested with a restriction enzyme resulting in DNA fragments of a specific size. The resulting DNA fragments are cloned into suitable vectors. These recombinant molecules are further transferred into the host cells to create a library. This library contains representative copies of all DNA fragments present within the genome. The main variable in constructing a genomic library is a type of vector used for the cloning of DNA fragments, which will determine the size of DNA fragments that can be cloned. Generally, high capacity cloning vectors are used for the construction of genomic libraries. Various high capacity cloning vectors such as l replacement vector, cosmid, yeast artificial chromosome (YAC), bacterial artificial chromosome (BAC), or P1 vector is used for cloning of DNA fragments

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Isolation of genomic DNA Partial restriction digestion Fragments of DNA

Clone the DNA fragments in a suitable vector

Selection of recombinant

Transfer the recombinant to the host

Genomic library

FIGURE 2.7 Construction of genomic library.

depending on the size of the fragment. Genomic libraries serve as a source of genomic sequence. It is used for genome mapping and genome sequencing purposes. It is also used to study the genetic mutation and function of regulatory sequences.

2.3.6 cDNA libraries cDNA libraries have been broadly used to determine the expressed portion of protein-coding genes in eukaryotes. The construction of a cDNA library involves the extraction and purification of mRNA (Fig. 2.8). These mRNAs are used as a template for the synthesis of cDNA by the process of reverse transcription in the presence of oligo dT primer. The oligo dT primer binds with the poly-A tail of mRNA followed by synthesis of the first strand of cDNA by using reverse transcriptase enzyme.

Isolate mRNA Reverse transcriptase Synthesis of first strand of cDNA by reverse transcription using oligo dT primer

cDNA amplification for second strand synthesis

Clone the cDNA in a suitable vector

Transfer the recombinant vector to the host

cDNA library FIGURE 2.8 Construction of cDNA library.

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After the synthesis of the first strand, mRNA is removed from DNA: RNA hybrid with the help of RNAse enzyme leaving a single-stranded cDNA. This single-stranded cDNA has the tendency to form a hairpin loop at the 3ʹ end, which provide 30 hydroxyl group for second-strand synthesis (self-priming). The single-stranded cDNA is converted into a double-stranded DNA with the help of DNA polymerase. After the synthesis of the second strand, the loop at 30 end is opened by the action of single-strand-specific S1 nuclease. The synthesized cDNA is further cloned into a suitable vector, followed by the transformation of recombinant vectors into a suitable host to create a cDNA library. cDNA libraries contain only the actively transcribed genes of an organism. The cDNA libraries lack information about enhancers, introns, and other regulatory elements because cDNA is synthesized from fully transcribed and processed mRNA. Introns would pose a problem when the eukaryotic gene is expressed in bacteria because most bacteria do not have any mechanism for the removal of introns. cDNA can be promptly expressed in a bacterial cell because mature mRNA is already spliced in eukaryotic cells, and hence the produced cDNA lacks introns.

2.4 Genomics Genomics is the study of structure, function, and interrelationships of both individual genes and the genome (Bazer and Spencer, 2005). This field has evolved from identifying small nucleotide segments of DNA from the sequencing of an organism’s complete genome. According to the Food and Agriculture Organization of the United Nations (FAO), by the year 2050, the global population will reach 10 billion, while the economic condition of the population in developing countries will continue to improve, which will lead to increased demand of animal products. Increasing animal production needs a deeper knowledge of animal biology through genomics and other relevant sciences. The livestock, poultry, and aquaculture require enhanced production and maintain global competitiveness with reduced greenhouse gas emissions.

2.4.1 Types of genomics Animal (livestock) genomics can be divided into structural genomics (the genome sequence and its variations), functional genomics (how the sequence is expressed), and comparative genomics (differences between different organisms at genome level).

2.4.1.1 Structural Structural genomics is an effort to depict a three-dimensional structure of every protein via experimental or computational approaches or a combination of both. Initially, the determination of the 3-D structures of proteins was based on curiosity or hypothesis-driven research. Determination of structures is so important because they tell us something new about biological processes such as the nature of a molecular recognition process, details of an enzyme mechanism, or about the energy transduction processes. The most important development of structural biology is the breakthrough of new relationships between protein structures and amino acid sequences. In view of this, new computational tools are required to be developed for concepts such as fold, protein family, and superfamily (Orengo et al., 1997; Hubbard et al., 1999) that help us to understand the complex 3-D structure of proteins. Various step involved in genomics are as follows: (i) Construction of high resolution genetic and physical maps (ii) Sequencing of the genome (iii) Determination of the complete set of proteins in an organism The sequencing of the genome is achieved by two methods, i.e., clone by clone sequencing and shotgun sequencing. In the clone by clone method, the fragments are first aligned into contigs. These fragments are then used to create cosmid and plasmid clones. Each clone of the contig is then sequenced. In shotgun sequencing, randomly selected clones are sequenced until all clones in the genomic library are assessed. Assembler software organizes the nucleotide sequence information so obtained into a genome sequence.

2.4.1.2 Functional Functional genomics is the study of how genes and intergenic segments of the genome contribute to different metabolic pathways (gene expression pattern). The main objective of functional genomics is to resolve how the individual segment of an organism work together to produce a particular phenotype. It relies on the dynamic expression of gene products in a definite background such as during a disease or at a specific developmental stage. Thus functional genomics involved in the development of a model link between genotype to phenotype. Functional genomics focused on at several levels such as

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DNA (genomics and epigenomics), RNA (transcriptomics), protein (proteomics), and metabolite (metabolomics). The data generated in it are subdivided into sequence and experimental datasets. The sequences are useful in fundamental genetic analysis, for example, SNP detection, a homology search, nucleotide substitutions, gene expression level, nucleotide composition analysis, and gene structure, etc. These fundamental methods commonly rely on genome-based sequence datasets using automated algorithms running in silico; for example, the function and functional interactions of unknown open reading frames (ORFs) can be predicted by using the principle of conserved operons (Overbeek et al., 1999). DNA microarrays and other high-throughput techniques, for instance, those involved in the investigation of proteins and metabolites, are considered as a major tool that initiated the establishment of the functional genomics.

2.4.1.3 Comparative As on Jan 25, 2007, 472 genomes of various organisms were completely sequenced, and yet another 498 are in progress (Sivashankari and Shanmughavel, 2007). The rapid progress in genome sequencing needs more proportional analysis to gain new insights into biochemical, evolutionary, genetic, physiological, and metabolic pathways. Comparative genomics is the science that deals with the comparison of the genetic material of one individual against that of another to add an improved understanding of how species evolved and to decide the function of coding and junk regions in genomes. The comparison may consist of gene number, gene content, the length, gene location, and the number of exons within genes, the amount of intron in each genome, and conserved sequences present in both prokaryotes and eukaryotes. By comparing DNA and protein sequences between species or among populations within a species, we can guess the rates at which different sequences have evolved and gathered chromosomal rearrangements, duplications and deletions. Comparative genomics not only reveals an evolutionary relationship between organisms but also similarities and dissimilarity between and within the species. For the evaluation of the unique features of humans, the most feasible study involves comparing humans to the chimpanzees and apes, which are our closest relatives. Sequence comparisons are necessary for forecasting the role that is to be played by a particular functional region, e.g., coding for a protein or regulating the level of expression of a gene. Information that arises from comparative genomics has a strong impact on medical genetics. As more and more loci are assigned to be responsible for disease and susceptibility to diseases, identification of the reason for the causative mutations becomes more difficult. Fitch (1970) developed a method called BBH (Best Bidirectional Hits), which discover best match individual gene pairs as orthologous. Tatusov et al. (2001) further improved the Fitch method, which matches groups of genes to groups of genes. In order to compare the genome of different organisms, it is necessary to understand the orthologues and paralogues. Orthologues are the homologous genes present in different organisms, but they encode proteins having a similar function. They have originated by direct vertical descent and have diverged simply by accumulating mutations. Paralogues are homologous genes present within the same organisms. These genes encode proteins having nonidentical functions. These genes originated by gene duplication followed by mutation accumulation. 2.4.1.3.1 Exon shuffling Although introns are noncoding sequences the most important role of introns in the evolution of genomes is the exon shuffling. Exon shuffling is the nonhomologous rearrangements between genes. The bulkier size of introns is more prone to random rearrangements within them and brings exons into new combinations with much higher frequency than would be possible for rearrangements in exons (the coding sequences). Sometimes these changes are the culprit for the high frequencies of deleterious rearrangements in large genes. The genetic diseases, for example, familial hypercholesterolemia and muscular dystrophy, are the result of these rearrangements. Both secreted and membrane proteins possess many extracellular protein domains that may be encoded by single exons. These extracellular protein domains arise due to extensive shuffling in exons during evolution. Exon shuffling is a molecular mechanism to produces new genes that encode proteins with altered functions. These proteins are called mosaic proteins, for example, serine proteases of blood coagulation. All members of the Ig superfamily, growth factor receptors, and cell adhesion molecules also possess this arrangement. The intervening sequences separating Ig type domains are always present between the first and second nucleotides of a codon (phase 1 intron). The Ig type domain is only one of at least seven types of domains that are characteristically encoded by phase 1 intron and that appear in different genes in a variety of combinations. The Ig- type domains are found in many adhesion molecules in tandem with domains distantly related to the type III repeats of fibronectin. Epidermal growth factor, lectin domains, and fibronectin type I and II repeats are some other examples of widespread phase 1 domain families.

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2.4.1.3.2 Genome similarity The similarity of related genomes is the basis of comparative genomics. Comparative analysis of the genome of different organisms having a recent common ancestor reveals that they may differ remarkably in appearance but may be quite similar at the genetic level. These differences between genomes are evolved from the ancestors’ genome during the course of evolution, and the closer relationship between the two organisms is responsible for higher similarities between their genomes. The calculation of DNA sequence similarity is a tough task. World average genetic difference or average sequence identity between any two species depends on the types of DNA sequences that are included in the calculation. Humans are most closely related to the great apes (orangutans, bonobos, gorillas, and chimpanzees) of the family Hominidae. On sequence comparison, it was found that humans and chimpanzees are about 1.1%e1.4% different at the level of whole-genome DNA sequence (Chen and Li, 2001; Ebersberger et al., 2002) thus humans share about 98.8% of their DNA with bonobos and chimpanzees. Similarly, humans also share their DNA sequence with gorillas, orangutans, and monkeys about up to 98.4%, 96.9%, and 93%, respectively. Not only the members of Hominidae family, but the human DNA sequence is also comparable with other unrelated species such as mice, dogs, and chicken, etc. It is believed that the human and mice diverged from the common ancestor some 100 million years ago. During this period, their functional DNA has diverged only to a limited extent, whereas their noncoding sequences have diverged to a great extent. Humans and mice share nearly 90% of their DNA, and it is beneficial because mice are used in laboratories as experimental model animals for research of human diseases. Presently mice are used in genetic research to study gene therapy and gene replacement. The similarity of DNA sequence between humans and dogs is about 84%, which is again useful for the study of human diseases, especially that are common for both organisms such as retinal disease, retinitis pigmentosa, cataracts, epilepsy, and allergies, etc. 2.4.1.3.3 Gene order comparison Comparing gene order in dissimilar organisms is one of the tools for developing molecular phylogeny. When gene order in a given region of the two organisms is comparable, they are termed as syntenic, and the phenomenon is called synteny. Synteny is the distribution pattern of genes on a chromosome. This pattern of gene locations in evolutionarily related species can be conserved in such a way that genes positioned near each other on the genome in one species are probably found close to each other on a single chromosome. A comparison of gene order in between two organisms is necessary to reveals many cases of inversions, duplication, insertion, and deletion of bases. Inversions in prokaryote involve a large segment of genomes and are mainly found at the origin and terminus of replication. Eukaryotes show a higher rate of inversion in comparison to prokaryotes as they have a larger genome. Some regions of the human genome are highly conserved in dogs, cattle, and sheep. 2.4.1.3.4 Horizontal gene transfer Horizontal or lateral gene transfer is the genetic exchange between different evolutionary lineages. It is believed that these transfers are common in the course of evolution and allows for the gaining of novel traits that are unique from those inherited. Horizontal gene transfer (HGT) has emerged as an important evolutionary tool for the evolution of prokaryote genomes, and as a result, in the evolution of Bacteria and Archaea domains (Boto, 2010; Syvanen, 2012). However, the importance of this process in eukaryotic evolution is not clear (except for gene transfers from mitochondria and plastid ancestors to the eukaryotic nucleus (Keeling and Palmer, 2008). Initially, the importance of this event in eukaryotic genomes evolution was not considered, but now the researchers are starting to acknowledge its importance in the evolution of unicellular eukaryotes (Tucker, 2013). The large-scale genome sequencing has improved our knowledge about the significance of HGT, especially in Eubacteria. The phylogenetic study of 144 prokaryotic genomes pointed out that most of the genetic information flow was vertical, but genes are also normally transferred horizontally between closely related taxa and between bacteria residing in the same environment (Beiko et al., 2005). HGT in Eubacteria is important to acquire many evolutionary traits such as pathogenesis, drug resistance, and bioremediation (Boucher et al., 2003). HGT among eukaryotic organisms is a very less common event. In animals, the eukaryote to eukaryote HGT event consists of the attainment of P elements by Drosophila melanogaster from Drosophila willistoni (Daniels et al., 1990), transfer of genes for carotenoid biosynthesis from fungi to pea aphids (Moran and Jarvik, 2010) and lectin-like antifreeze proteins between fishes (Graham et al., 2008). Recently, heritable HGT was discovered in humans from the mitochondrial-derived minicircles in the Trypanosoma cruzi (Hecht et al., 2010), suggesting that HGT can occur in human germ cells. Most of the HGT events described were found within a single domain of life and mainly involved bacteria to bacteria transfers. HGT event between the different domains of life (Archaea, Eubacteria, and Eukaryota) has also been explained; for example, the Eubacteria Thermotoga

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maritima harbor 81 archaeal genes clustered in 15 4e20 kbp islands (Nelson et al., 1999). One of the best-studied examples involving interkingdom gene transfer between Eubacteria and Eukaryota is the Agrobacterium tumefaciens that naturally transfers T-DNA from its Ti plasmid to plants (Gelvin, 2003). There are the following two methods that are used for the detection of genes, which are acquired by HGT: (i) Detection of genes having an unusual base composition (ii) Failure to find a similar gene in closely related species The availability of complete genome sequences has facilitated the detection of such genes. 2.4.1.3.5 Single nucleotide polymorphisms (SNPs) Molecular markers are the polymorphisms revealing tools at the DNA level and are now playing an important role in animal genetics. There are three types of variation at the DNA level in population: (i) a single nucleotide difference known as SNPs for single nucleotide polymorphisms (ii) insertions or deletions of various lengths ranging sequences from one to several hundred base pairs and (iii) variations in the number of tandem repeats (VNTR). If we consider molecular markers in terms of the type of information they give at a single locus, there are only three main categories that are described as follows: (i) The biallelic dominant marker such as AFLPs (amplified fragment length polymorphisms) and RAPDs (random amplification of polymorphic DNA) (ii) The biallelic codominant marker such as SSCPs (single-stranded conformation polymorphisms) and RFLPs (restriction fragment length polymorphisms) (iii) The multiallelic codominant marker such as the microsatellites SNP (single nucleotide polymorphism) is the single base positions in genomic DNA at which different nucleotides occur in different individuals of a population. Each nucleotide at such a position is referred to as an allele of the SNP. The least frequency of the SNP allele is 1% or more in the genomic DNA of individuals. Although at each position of a particular DNA sequence, any of the four possible nucleotide bases can be present, but SNPs are usually common in biallelic structure. Several different methods have been developed to discover SNPs. A simple procedure used to analyze the stored sequence data from the database to identify SNPs is a DNA chip or Microarray. The lowest frequency of single nucleotide substitutions at the origin of SNPs in mammalian DNA was estimated between 1  109 and 5  109 per nucleotide and per year at neutral positions (Martinez-Arias et al., 2001). It is estimated that 90% of sequence variation in humans is attributed to SNPs. The human genome contains about 3e17 million SNPs. Thus every gene may be expected to contain w6 SNPs. 2.4.1.3.6 Phylogenetic footprinting The comparative analysis of genome sequences of related species to detect orthologous DNA sequences is known as phylogenetic footprinting. There are tools that predict the regions of the genome that correspond to protein-coding genes, but they are less efficient in finding out the parts of the genome that are being transcribed. Phylogenetic footprinting is a useful tool for describing regions of functional importance in genomic sequences. Also, this technique has been used to estimate the number of protein-coding genes that expose genes or exons that had not been demonstrated by current gene prediction programs (Crollius et al., 2000; Gilligan et al., 2002). Further, it has also displayed a high degree of conservation that is found in regions outside the exons, corresponding to nonprotein coding transcribed sequences and regions of regulatory importance (Bejerano et al., 2004).

2.5 Evolution of animal genomics The term “genome” was coined by Hans Winkler in 1920 by merging the words gene and chromosome. Later on, this concept was challenged by Lederberg and McCray, who suggested that Winkler probably merged gene with the generalized suffix ome, which means “the entire collectivity of units,” and not -some (“body”) from the chromosome. Genome is the complete set of all genes, which stores the biological information. The nature of the genome may be DNA or RNA. All eukaryotes and prokaryotes always have a DNA genome, but the virus may either have a DNA genome or RNA genome. The eukaryotic genome consists of two distinct parts: The nuclear genome and organelle (mitochondria and chloroplast) genome. The amount of DNA present in the genome of a species is called C-value, and the size of the genome

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represents one of the most important parameters to understand the complexity in eukaryotic organisms. Genomic comparisons of different organisms clearly indicate that the complexity of the organism tends to increase with the genome size. Genomes sizes of closely related species often show remarkable variation in size (Gregory, 2005; Bennett and Leitch, 2011). Genomes can even vary between cells within individuals due to programmed genome rearrangement and somatic mutation (Smith et al., 2012). Many factors can influence the evolution of genome size by genome expansion and contraction, but the evolutionary processes that dominate remain mostly unresolved. However, continuous approaches toward the appropriate causes of genome size evolution have been made to characterize the process. As genome sequences become available from a greater number of species, the phylogenetic comparative method is now becoming useful to detect the phenotypic differences during the evolution of the genome (Felsenstein, 2008). Phenotype changes take place with changes in the genome along each branch of the animal tree, but it does not mean that phenotypic changes will always appear with a change in the genome. Due to the complicated nature of the animal genome, it is very difficult to identify genes specific to phenotypic novelties (Zhang et al., 2013; Kapheim et al., 2015). Phenotypic differences may also occur due to genome rearrangements, such as inversion, translocations, duplication, fission, and fusion. Genomic rearrangement polymorphisms in eukaryotes, along with the different environmental conditions, cause speciation, but there is little evidence about it (Pinton et al., 2003; Coghlan et al., 2005). Comparative genomic analysis of different domestic breeds provides an efficient way of exploiting the genetic basis of phenotypic variation (Andersson and Georges, 2004). But, the role of genome size in the evolution of organismal complexity remains unanswered. In lower eukaryotic organisms, the amount of DNA increases with increasing complexity. However, in higher eukaryotes, there is no correlation between increased genome size and complexity, which is referred to as the C-value paradox. The C-value (genome size) paradox clearly suggests that a larger eukaryotic genome size does not correlate closely with organismal complexity (Gregory, 2005). For example, a man is more complex than amphibians in terms of genetic development, but some amphibian’s cells contain 30 times more DNA than the human cells. However, more complex organisms have big size genomes as compared to the less complex organism that has small size genomes. There is no single axis of organism complexity, and each animal has a mix of traits that could be characterized as simple or complex (Dunn and Ryan, 2015). The complete genome of Drosophila melanogaster contains 21 nuclear receptors, compared to 49 in the human genome (Garcia et al., 2003). Current genomic and evolutionary research on nonhuman animals will be helpful for a better understanding of the biology and evolution of animal genome within the animal kingdom (Song and Wang, 2013).

2.5.1 Mapping genomes The ultimate aim of genomics is to obtain the DNA sequence of the complete genome, which provides the most detailed molecular description, i.e., the complete nucleotide sequence of the genome. Genome mapping of an organism is an important tool to provide the exact positions of genes in the chromosomal DNA. Distinctive features such as restriction fragment length polymorphism (RFLPs), simple sequence length polymorphism (SSLPs), and single nucleotide polymorphisms (SNPs) on the genome map are used as landmarks to construct the genome map. A complete physical-genetic map of the genome is necessary for the manipulation of genes in various cloning applications that relies on mapping close to a convenient marker. For understanding the animal evolutionary history and genetic diversity, a variety of genetic markers can be utilized and grouped into two types. Type I markers are DNA segments with a low degree of polymorphism but high evolutionary conservation and encodes protein synthesis, whereas Type II markers are DNA segments with high polymorphism, not well conserved and do not have any identifiable biological function (O’Brien, 1991; Dodgson et al., 1997; Morin et al., 2004). Traditionally, Genome mapping has been done by using two approaches: genetic mapping or physical mapping (Murphy et al., 2001).

2.5.1.1 Genetic mapping Genetic mapping is used to identify the exact position of a gene on the particular chromosome. It also provides information about the recombination based on the distance between the genes. The resolution of the genetic map depends upon the number of crossovers in a large number of progenies in humans and other eukaryotes. There are three types of DNA markers that are useful for genetic mapping, namely, restriction fragment length polymorphism (RFLP), simple sequence length polymorphisms (SSLPs), and single nucleotide polymorphisms (SNPs). SNPs are single base pair positions and most important sequence markers for mapping of genomes. Maps based on sequence-tagged site (STS)

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landmarks provide greater coverage of the genome (Bouffard et al., 1997). STS is 200e300 bases long, and a unique DNA sequence appears only singly in the genome. The partial DNA sequences termed expressed sequence tags (ESTs) are easier to generate and serve the same purpose as genomic STS (Harushima et al., 1998).

2.5.1.2 Physical mapping For the physical map construction, mostly molecular techniques are required. A physical map of an organism contains overlapping portions of the genome. The physical mapping starts from the formation of cloned genomic library fragments, normally prepared by either random mechanical breakage or partial restriction digestion of genomic DNA. DNA fragments are usually cloned in bacterial hosts using plasmid, bacteriophage, cosmid, or other vector systems. For the physical mapping of large genomes, bacterial artificial chromosomes or P1 artificial chromosomes are used to insert the more than 100 kb DNA fragment. Even larger fragments (over 1Mb) can be cloned in yeast (Saccharomyces cerevisiae) using yeast artificial chromosome (YAC) vectors; however, such clones are often unstable, undergoing deletions or internal rearrangements (Dear, 2001). Genetic maps based on variations in simple sequence repeats also enable the generation of highly detailed genetic maps (Dib et al., 1996).

2.5.2 Regulation of gene expression Gene regulation is the cellular processes that control the rate and pattern of gene expression. The interactions between genes, regulatory proteins, and other components involved in expression determine when, where, and how many specific genes are expressed. All the genes of any organism are not expressed at the same time. Most of the genes of an organism are expressed during a particular stage of development. Some genes are continuously expressed as they produce proteins involved in basic metabolic functions and these are called housekeeping genes. Some genes are expressed as part of the cellular process called the facultative gene. Gene expression can be regulated with the help of various regulatory proteins during cellular processes at numerous levels. These regulatory proteins bind to the regulatory region of DNA and send signals that indirectly control the rate of gene expression. Regulatory protein or factors have two main functional domains; the first DNA binding domain that enable them to bind to the specific response elements and second activation domain that interacts with or binds to the other components of the transcription apparatus. The up-regulation of a gene refers to an increase in expression of a gene, while downregulation refers to the decrease in expression of a gene and corresponding protein expression. The upregulation process occurs within a cell, and gene expression is triggered either by an internal or external signal. Increased expression of one or more genes results in increased proteins encoded by those genes. In this case, more receptor protein is synthesized and transported to the membrane of the cell, and thus, the sensitivity of the cell is brought back to normal, reestablishing homeostasis.

2.5.2.1 Regulation of gene expression in eukaryotes The gene expression in eukaryotes is more tightly regulated as compared to gene expression regulation in prokaryotes. In eukaryotes, genetic material and translation machinery are separated by a nuclear membrane. Regulations of gene expression in eukaryotes take place at various stages, as explained below. 2.5.2.1.1 Chromatin structure In mammalian cells, DNA is packaged into a nucleoprotein complex called chromatin. Nucleoproteins are mainly of two kinds, i.e., histone and nonhistone proteins. So, chromatin has mainly three components: DNA, histone, and nonhistone protein. Earlier evidence suggests that histone may be involved in repressing gene activity, but later on, specific regulation by nonhistone protein was also demonstrated, which suggests that the mRNA synthesized under in vitro condition from reconstituted chromatin, mainly depends on the source of nonhistone protein. In recent years, DNA supercoiling (SC) attracted considerable attention for its role in gene regulation. Due to the helical nature of DNA, transcription at both initiation and elongation steps are regulated in eukaryotes (Leblanc et al., 2000; Hatfield and Benham, 2002; Travers and Muskhelishvili, 2005). The environmental response causes fast changes in DNA topology, which play an important role in the regulation of gene expression (Ouafa et al., 2012; Dorman and Dorman, 2016). Mutational experiments with bacteria, which cause the unfolding of supercoiling structure, show the role of supercoil structure in genome evolution (Crozat et al., 2005).

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2.5.2.1.2 Initiation of transcription Gene expression can be modulated by promoters, enhancers and other regulatory factors. Two levels of gene expression are present in eukaryotes. The first level of regulation occurs at the transcription initiation, and the second level occurs at the posttranslational level. It has the ability to alter the binding of RNA polymerase with the DNA, and hence, regulate the initiation of transcription. Enhancers are much more common in eukaryotes than prokaryotes (Austin and Dixon, 1992). Silencers are specific DNA sequences, and when particular transcription factors bind with these specific DNA sequences, they can decrease the expression of the gene. The most important and diverse mechanisms of gene regulation in both prokaryotic and eukaryotic cells are binding of transcriptional factors at a sequence-specific region of the DNA (Pulverer, 2005). In eukaryotes, regulation of gene expression by transcription factors is said to be combinatorial, in which case, it requires the coordinated interactions of multiple proteins in comparison to prokaryotes where usually single protein is required (Phillips, 2008). 2.5.2.1.3 Post-transcriptional processing Post-transcriptional regulation may occur at the level of RNA processing, RNA transport, and post-transcriptional modifications. Proteins that may be involved in the regulation of RNA processing are the protein-containing ribonucleoprotein (RNP) domains. RNPs have an important function in post-transcriptional regulation of gene expression. During RNA processing, nearly 60% of all genes may be alternatively processed to generate a much greater diversity of proteins to compensate for the relatively small number of genes in the genome. Modifications such as polyadenylation, capping, and different splicing patterns of the pre-mRNA transcript in eukaryotes can lead to different levels of gene expression. The stability of eukaryotic mRNAs in the cytoplasm also regulates the gene expression in eukaryotes. Mature mRNAs are transported from the nucleus to the cytosol for translation into a protein. This is the main point of regulation of gene expression in eukaryotes. 2.5.2.1.4 Initiation of translation mRNA contains a large number of RNA binding proteins sequences that direct the translation initiation. The binding of the regulatory protein to their target sequence on mRNA is controlled by the secondary structure of the transcript, which depends on certain conditions, such as temperature or presence of a ligand. There are a number of mechanisms that are controlled at the level of translation initiation. For starting the translation, it is necessary to bind a protein called eukaryotic initiation factor-2 (eIF), which must bind to a part of the ribosome called the small subunit. Phosphorylation or addition of a phosphate group to the eIF-2 regulates the binding of eIF-2 to the regulatory sequences. Binding of the small ribosomal subunit to the mRNA can be modulated by means of mRNA secondary structure, antisense RNA binding, or protein binding. More than 25 proteins are needed for proper translational initiation as compared to elongation and termination, where only a few proteins are needed (Preiss and Hentze, 2003; Pestova et al., 2007). 2.5.2.1.5 Post-translational processing The translated proteins go for the post-translational modification that includes glycosylation, fatty acylation, and acetylation modifications in polypeptide chains. These modifications also regulate the expression of the gene. After translation and processing, proteins must be transported to their site of action in order to be biologically active. Gene expression can also be controlled with the stability of proteins, which greatly depend on specific amino acid sequences and composition present in the proteins (Mata et al., 2005).

2.5.2.2 Regulation of gene expression in prokaryotes Prokaryotic genes are organized in groups called operons, each of which code for a corresponding protein. An operon consists of a structural gene whose transcription is regulated by the same sets of genes i.e., regulator gene, promoter, and operator gene. An operon is regulated by specific proteins that bind to the DNA at the operator region. These proteins are called regulatory proteins. It is chiefly controlled by two DNA sequence elements of size 35 and 10 bases, respectively. There are two major modes of transcriptional control in E. coli to modulate gene expression, as described below. 2.5.2.2.1 Catabolite-regulation Catabolite sensitive operons are repressed by accumulation breakdown products (catabolites) of various carbon compounds. The lac operon present in E. coli is an example of catabolite regulation. The lac operon is an inducible operon that utilizes lactose as an energy source and is activated when glucose is low, and lactose is present. The presence of glucose in

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medium has a positive effect on the expression of genes involved in the catabolism of alternative sources of carbon such as lactose. When glucose is present in the medium, genes related to catabolism of other energy sources are not expressed. The presence of glucose represses the lac operon even if an inducer (lactose) is present. 2.5.2.2.2 Transcriptional attenuation Transcription units subjected to attenuation contain attenuator. An attenuator is an intrinsic terminator located at the beginning of a transcription unit. The phenomena of attenuation control the ability of RNA polymerase to read through an attenuator. When RNA polymerase continues transcription, attenuation works by controlling the formation of the hairpin loop. The formation of the hairpin loop depends on the availability/nonavailability of specific amino acids. If the concerned amino acid is not available, the hairpin loop does not form in the RNA transcript at attenuator, and as a result, transcription continues. However, if a concerned amino acid is available, the hairpin loop is formed at an attenuator. As a consequence, transcription stops before it reaches the first structural gene of the operon. Trp operon of E. coli is an example of an attenuated operon that encodes five enzymes necessary for tryptophan biosynthesis. When tryptophan is not available in medium, then only genes related to tryptophan biosynthesis are expressed.

2.6 Role of genomics in animal improvement As the world population increases, the demand for livestock products is likely to increase in the coming decades (Delgado et al., 1999). Genomics can play a major role in sustainable food security by expanding the utility, diversity, and yield of resources. The utilization of genomics in animal genetic improvement could accomplish the increased efficiency desired. The new high throughput genomics tools are used to improve the genetic diversity, quality, and safety of livestock for sustainable agriculture. The genomic technologies will expand the endeavors to recognize the genes and genetic mechanisms underlying economically significant characteristics in livestock species. The major role of genomics in livestock improvement and production is the identification of superior parents, marker-assisted selection, marker-assisted introgression, and genomic selection. The polymorphic markers are associated with economically significant characteristics, which are very useful in animal breeding. The genetically superior animals are used as parents for subsequent generations in livestock breeding programs. Genomic technologies are also enhancing the genetic variation, selection precision, selection intensity, and diminishing the generation interval. Genomics provides the genome information, which is very useful for the improvement of animals. It helps in the sustainable production and preservation of genetic resources. Wholegenome sequencing has made a significant contribution to the generation of genomic information (Druet et al., 2014). The chicken genome was the first livestock to be sequenced in 2004 (Groenen et al., 2009), followed by the sheep, cattle, pig, and goat genomes in 2007, 2009, and 2013, respectively (Fan et al., 2010). The role of genomics in improving animal health will become increasingly important. The mapping of animal genomes with the help of genomics is very useful in the identification of susceptible and resistant genes to improve animal health. Genomics is an attractive way to improve the overall health of animals and to reduce the effect of infection by various pathogens.

2.7 Conclusions Genomics is the study of structure, function, and interrelationships of both individual genes and the whole genome. This field has advanced from identifying short nucleotide sequence of DNA to the sequencing of the entire genome of an organism. The genome sequencing work provides an enormous quantity of new information that represent molecular blueprints of a number of organisms from microbes to humans. Genomic sequence information is precious for characterizing evolutionary and functional relationships between related genes. This information is also used for the identification of gene products that are involved in human diseases. Genomics approaches in farm animals present a key prospect to address the responsibilities of agricultural production for society at an economic level. They may also contribute to environmental sustainability, the source of healthier human foods, and human medicines. The important role of genomics in animal improvement is the identification of superior parents, marker-assisted selection, marker-assisted introgression, and genomic selection.

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Pestova, T.V., Lorsch, J.R., Hellen, C.U.T., 2007. The mechanism of translational initiation in eukaryotes. In: Mathews, M.B., Sonenberg, N., Hershey, J.W.B. (Eds.), Translational Control in Biology and Medicine. Cold Spring Harbor Laboratory Press, New York, pp. 87e128. Phillips, T., 2008. Regulation of transcription and gene expression in eukaryotes. Nat. Edu. 1 (1), 199. Pinton, A., Ducos, A., Yerle, M., 2003. Chromosomal rearrangements in cattle and pigs revealed by chromosome microdissection and chromosome painting. Genet. Sel. Evol. 35 (6), 685e696. Polyak, K., Meyerson, M., 2003. Overview: Gene Structure. Cancer Medicine, 6 ed. BC Decker. Porreca, J.G., 2010. Genome sequencing on nanoballs. Nat. Biotechnol. 28 (1), 43e44. Preiss, T., Hentze, M.W., 2003. Starting the protein synthesis machine: eukaryotic translation initiation. Bioessays 25, 1201e1211. Pulverer, B., 2005. Sequence-specific DNA-binding transcription factors. Nat. Milest. https://doi.org/10.1038/nrm1800. Ridgway, P., Almouzni, G., 2001. Chromatin assembly and organization. J. Cell Sci. 114, 2711e2712. Russell, P., 2002. iGenetics. Pearson Education, Inc., San Francisco, pp. 187e189. Salgado, H., Moreno-Hagelsieb, G., Smith, T., Collado-Vides, J., 2000. Operons in Escherichia coli: genomic analyses and predictions. Proc. Natl. Acad. Sci. U. S. A. 97 (12), 6652e6657. Schaefer, G.B., Thompson, J.N., 2014. Medical Genetics: An Integrated Approach. Mcgraw hill, 1/E edition. Schumperli, D., McKenney, K., Sobieski, D.A., Rosenberg, M., 1982. Translational coupling at an intercistronic boundary of the Escherichia coli galactose operon. Cell 30 (3), 865e871. Shafee, T., Lowe, R., 2017. Eukaryotic and prokaryotic gene structure. Wiki. J. Med. 4 (1), 2. Shizuya, H., Birren, B., Kim, U.J., Mancino, V., Slepak, T., Tachiiri, Y., Simon, M., 1992. Cloning and stable maintenance of 300-kilobase-pair fragments of human DNA in Escherichia coli using an F-factor-based vector. Proc. Natl. Acad. Sci. U. S. A. 89 (18), 8794e8797. Sivashankari, S., Shanmughavel, P., 2007. Comparative genomics - a perspective. Bioinformation 1 (9), 376e378. Smith, J.J., Baker, C., Eichler, E.E., Amemiya, C.T., 2012. Genetic consequences of programmed genome rearrangement. Curr. Biol. 22, 1524e1529. Song, L., Wang, W., 2013. Genomes and evolutionary genomics of animals. Curr. Zool. 59 (1), 87e98. Struhl, K., 1999. Fundamentally different logic of gene regulation in eukaryotes and prokaryotes. Cell 98 (1), 1e4. Syvanen, M., 2012. Evolutionary implications of horizontal gene transfer. Annu. Rev. Genet. 46, 341e358. Tatusov, R.L., Natale, D.A., Garkavtsev, I.V., Tatusova, T.A., Shankavaram, U.T., Rao, B.S., Kiryutin, B., Galperin, M.Y., Fedorova, N.D., Koonin, E.V., 2001. The COG database: new developments in phylogenetic classification of proteins from complete genomes. Nucleic Acids Res. 29, 22e28. Thomas, M.C., Chiang, C.M., 2008. The general transcription machinery and general cofactors. Crit. Rev. Biochem. Mol. Biol. 41 (3), 105e178. Tian, T., Salis, H.M., 2015. A predictive biophysical model of translational coupling to coordinate and control protein expression in bacterial operons. Nucleic Acids Res. 43 (14), 7137e7151. Travers, A., Muskhelishvili, G., 2005. Bacterial chromatin. Curr. Opin. Genet. Dev. 15, 507e514. Tucker, R.P., 2013. Horizontal gene transfer in choanoflagellates. J. Exp. Zool. B Mol. Dev. Evol. 320B, 1e9. Venter, J.C., Adams, M.D., Myers, E.W., Peter, W.L., Mural, R.J., Sutton, G.G., Smith, H.O., 2001. The sequence of the human genome. Science 291, 1304e1351. Watson, J.D., Crick, F.H., 1953. Molecular structure of nucleic acids; a structure for de-oxyribose nucleic acid. Nature 171, 737e738. Werner, F., Grohmann, D., 2011. Evolution of multisubunit RNA polymerases in the three domains of life. Nat. Rev. Microbiol. 9 (2), 85e98. Zhang, G., Cowled, C., Shi, Z., Huang, Z., Bishop-Lilly, K.A., Fang, X., Wynne, J.W., Xiong, Z., Baker, M.L., Zhao, W., 2013. Comparative analysis of bat genomes provides insight into the evolution of flight and immunity. Science 339, 456e460.

Chapter 3

Stem cells: a potential regenerative medicine for treatment of diseases Dhruba Malakar, Hruda Nanda Malik, Dinesh Kumar, Sikander Saini, Vishal Sharma, Samreen Fatima, Kamlesh Kumari Bajwa and Satish Kumar Animal Biotechnology Centre, National Dairy Research Institute, Karnal, Haryana, India

3.1 Introduction Stem cells are the unspecialized cells having the capacity to self-renew themselves, as well as to generate differentiated cells. Stem cells have the remarkable potential to develop into all 220 cell types in the body during early life and growth. In many tissues, they serve as an internal repair system throughout the life of a person or animal. When a stem cell divides, each new cell has the potential to either remain a stem cell or become another type of cell with a more specialized function, such as a muscle cell, a red blood cell, or a brain cell. The stem cell is a new herald for the treatment of incurable diseases like mastitis, endometritis, wounds, cancer, fracture in animals and paralysis, and wounds in dogs. The aim of the application of the stem cells was to treat diseases in livestock with mesenchymal stem cells (MSCs) for higher productivity of animals and improve the economic condition. MSCs were aseptically isolated and cultured in in vitro condition and cryopreserved in
196 C liquid nitrogen. All the cells characterized by different marker genes like CD9, CD29, CD44, CD71, CD73, CD99, and CD105 were expressed, whereas no expression was observed for CD11b, CD14, CD34, and CD45 markers and differentiated into osteogenic, chondrogenic, audiogenic, neurogenic lineages. Different diseases of animals like mastitis, endometritis, fracture, cancer, FMD wounds can be treated with MSCs, and all these animals cured completely and permanently. There may not be any side effects to these animals. In conclusion, MSCs therapy is the most potent, simple, cheap, regenerative medicine therapy for the treatment of many diseases to cure animals and may be used in human beings in the future. Stem Cells can be divided based on the potency.

3.1.1 Totipotent stem cells Totipotent stem cells can give rise to whole organisms to form 220 cell types found in the body, as well as extraembryonic cells that are formed in the placenta.

3.1.2 Pluripotent stem cells Pluripotent stem cells can give rise to all 220 cell types of the body only but unable to form the placenta.

3.1.3 Multipotent stem cells Multipotent stem cells can develop into a group of cell types in a particular lineage like hemopoietic cells or blood cells.

3.1.4 Oligopotent stem cells Oligopotent stem cells have the ability of progenitor cells to differentiate into a few cell types like lymphoid blood cells such as B lymphocyte and T lymphocyte cells, but not all blood cells types are like a red blood cell.

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3.1.5 Unipotent stem cells Unipotent Stem Cells can differentiate into one type of stem cell-like hepatoblastoma cells that can differentiate into hepatocyte cells only.

3.2 History of stem cells 3.2.1 Types of stem cells 3.2.1.1 Embryonic stem cells Embryonic stem cells (ES cells) that were first derived from mouse embryos (Evans and Kaufman, 1981) reveal a new technique for culturing the mouse embryos for the derivation of ES cells from these embryos. The human embryonic stem cells (ESCs) are derived from the inner cell mass of the blastocyst embryo (Thomson et al., 1998). ES cells are undifferentiated pluripotent stem cells derived from the inner cell mass of a blastocyst, which has the capability to self-renew indefinitely in an undifferentiated state and differentiate in any lineage of cells in the body. These properties of ES cells are maintained by symmetrical self-renewal, producing two identical stem cell daughters upon cell division. The generation of pluripotent cells from differentiated adult cells has vast therapeutic implications, particularly in the context of in vitro disease modeling, pharmaceutical screening, and cellular replacement therapies. After fertilization of human embryos that reach the blastocyst stage on 4e5 days and the inner cell mass consists of 120e150 cells in number. In livestock species, cattle and buffalo also produce blastocysts on 6e7 days of postfertilization. The inner cell mass can be easily isolated surgically or seeded by hatching blastocysts in culture medium (Fig. 3.1). Then the inner cell mass will grow in the stem cell culture medium. These cells divide very frequently due to a shortened G1 phase in the cell cycle system. Faster cell division allows the cells to quickly grow in numbers only but not their size, which is important for early embryo development after fertilization in humans and animals. The ESCs express different pluripotency molecular factors like Oct4, Sox2, and Nanog, and surface markers like SSEA1 SSEA3, SSEA4, Tra-1-60, and Tra-181, which play a vital role in transcriptionally regulating the ESC cell cycle. The ESCs are pluripotent stem cells that can give rise to all the 220 cell types derived from all the three germ layers: Endoderm, Ectoderm, and Mesoderm and germ cells. When the stem cells get the appropriate signals or specific chemicals into the medium, the ESCs can differentiate into the desired cell types like cardiomyocytes (Garg et al., 2012, Geijsen et al., 2004). In the present day, there is a serious ethical problem with ESCs production all over the world, as destroying a blastocyst is destroying life. Therefore, ESCs cannot be used for the treatment of diseases due to ethical problems in humans and animals. But currently, the culture of ESCs is presently focused heavily by the researchers on the therapeutic potential in clinical application in many laboratories, especially the treatment of more prevalent diabetes, cancer, and heart disease. There are other areas of study as a model of genetic disorders, genomic modification, and DNA repair mechanisms. The adverse effects of ESCs have also been reported in clinical studies such as tumors and unwanted immune responses.

FIGURE 3.1 Generation of embryonic stem cells from in vitro produced hatched blastocyst.

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3.2.1.2 Adult stem cells Pluripotent stem cells used for treatment are increasingly restricted in their lineage potential and production of tissuespecific multipotent stem cells, which can differentiate into different lineages. The adult stem cells are undifferentiated or unspecialized cells found in a differentiated (specialized) tissue of the body of human beings and animals. These are capable of self-renewal, undifferentiated, and multipotent character that participates in the regeneration of damaged tissues of the body and replenishment of dying cells or dead cells. Sources of adult stem cells are found not only in bone marrow but also obtained from different human and animal organs such as adipose tissue, cornea, and retina of the eye, the dental pulp of the tooth, liver, skin, bloodstream, synovium, as well as adult human testis, etc. Adult stem cells may be pluripotent or multipotent stem cells and used for the treatment of different diseases in humans and animals due to relaxing ethical problems of autogenic or allogeneic adult stem cells. MSCs can be isolated from the body tissues and different approaches to characterize these cells in in vitro conditions for the propagation of a large number of cells for the treatment of different diseases. There are specific guidelines and protocols for the characterization and meticulous use of these cells for treatment. International Society for Cellular Therapy has given minimal criteria to define MSCs. First, MSCs must be plastic-adherent in in vitro culture conditions. Second, Based on the minimal criteria of the International Society of Cellular Therapy (ISCT), human MSCs identified by adherence to plastic and expression of cell surface markers include CD29, CD44, CD90, CD49a-f, CD51, CD73 (SH3), CD105 (SH2), CD106, CD166, and Stro-1 and lack of expression of CD45, CD34, CD14 or CD11b, CD79a orCD19, and HLA-DR surface molecules. Third, MSC must differentiate to osteoblasts, adipocytes, and chondroblasts in in vitro conditions. MSCs have no immunogenic effect and could replace the damaged tissues.

3.2.1.3 Induced pluripotent stem cells (iPSCs) Induced pluripotent stem cells are presently the most important pluripotent stem cells which can be generated directly from a somatic cell using some genes like Oct4, Sox2, Klf4, and c-Myc. iPSCs are adult somatic cells that have been genetically reprogrammed using these genes to express expression to produce an embryonic pluripotent stem cell-like state for maintaining the defining properties of embryonic stem cells. Mouse iPSCs were first reported in 2006 using Oct4, Sox2, Klf4, and c-Myc genes to produce embryonic stem cells like cells (Takahashi and Yamanaka, 2006), and human iPSCs were first reported in late 2007 (Okita et al., 2007). Although these cells meet the defining criteria for pluripotent stem cells, embryonic stem cells differ in clinically significant ways. These cells are a new herald in the field of regenerative medicine and translational science as these cells can be produced indefinitely in in vitro cultural conditions and differentiated to all the 220 cell types in the body like cardiomyocytes, liver, kidney, neurons, pancreatic cells, etc. The self-renewal and pluripotency properties are regulated by an array of protein-coding genes, such as transcription factors and chromatin remodeling enzymes, in a core regulatory circuitry (Jaenisch and Young, 2008). This circuitry includes Oct4, Sox2, and Klf4, which form self-regulatory networks and control a wide range of downstream genes (Liu et al., 2008). Extensive studies have indicated that Oct4, Sox2, and Klf4 are required for ESC self-renewal and pluripotency. These iPS cells can be repaired by the damaged tissues or diseases of the animals, which is propagated from a single source of in vitro culture pluripotent stem cells. Generally, embryonic stem cells are produced from preimplantation stage blastocysts, which totally binds to destroy any life in the world and much controversy for the use of these embryonic stem cells in human beings and animals. However, the iPS cells are the alternative to embryonic stem cells for regenerative medicine and research work. As the iPSCs are produced directly from adult tissues of the patients, they are not destroying an embryo and use patient-matched stem cells as the adult somatic cells of the same individual, and the patients can have their pluripotent stem cell line in vitro culture conditions. These autologous cells are produced indefinitely and used for transplantation without any risk of immune rejection in their body. But the iPSCs are not yet in an application stage to use in the patient through therapeutic purposes that have been deemed to be safer. But presently, iPSCs are mostly utilized for drug discovery or testing and research on patient-specific diseases. Embryonic stem cells are not used for the treatment of diseases of animals and human beings as there is an ethical problem throughout the world, and the production of iPSCs is tedious and time-consuming, although there is not much ethical problem. Application of adult stem cells is easily used for the treatment of different diseases of animals as there are fewer ethical issues, easy to isolate and characterize from the body of the animal or human being. Here, it is used to adipose tissue-derived MSCs for the treatment of different diseases in animals and the method of their isolation and characterization of these MSCs.

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3.3 Materials and methods MSCs exist in all tissues and can be easily derived from bone marrow, umbilical cord, fat tissue, liver, intestine, lung. Among another source of MSCs, adipose tissue-derived mesenchymal stem cells are a prime source of cell therapy under its easy availability, enormous expandability, ease of isolation. The adipose tissues derived from adult MSCs were cultured for cattle, buffaloes, dogs, and mice for treatment of mastitis, metritis, and hoof wounds of cattle and buffaloes, tibial fracture of mice and wounds and paralysis of dogs (Malik et al., 2013; Bajwa et al., 2017; Malakar et al., 2019). Adipose tissues were collected from the fat pad of the tail head region, the rump site of cattle, buffalo, and dog by liposuction methods and goat from the slaughterhouse and transferred in transport medium containing antibiotic solution. The adipose tissues were minced into very small pieces and incubated in digestive media containing DMEM F/12%, 1% type 1 collagenase enzyme for 4 h under standard culture condition. The dissociated cells were filtered in a 41 mm filter to remove undigested fat tissue. The cell suspension was centrifuged at 1000 rpm for 10 min, and the pellet was seeded in a 25 cm2 culture flask in a growing medium under standard culture conditions (Fig. 3.2). Next passage, the cells were trypsinized at 80% confluence cells and centrifuged at 1000 rpm for 10 min and reseeded in 25 cm2 culture flask at density 2  105 cells/cm2 and large number MSCs can be produced in this culture system for the treatment of the diseases. MSCs can differentiate into osteoblastic, adipocytic, neurocyte, and chondrocyte lineages (Fig. 3.3).

3.3.1 Cryopreservation of mesenchymal stem cells for a long time for further use The adipose tissues derived from in vitro cultured MSCs at 80% confluence cells were trypsinized and centrifuged at 1000 rpm for 10 min and mixed with cryopreserved medium with 10% DMSO and serum at density 1  106 cells and put into 2 mL cryovials. The cryovials were kept at 4 C for 4 h and
80 C overnight. Then the cryovials were shifted into a liquid nitrogen tank for further use. The cryopreserved cells were thawed at 37 C water bath and removed from the cryopreserved medium with centrifugation at 1000 rpm for 5 min. The pellet cells were further in vitro cultured for 3 days.

3.3.2 Characterization of adipose tissue-derived mesenchymal stem cells MSCs were characterized by alkaline phosphatase; different molecular markers like CD9, CD29, CD44, CD71, CD73, CD99, and CD105 were expressed whereas no expression was observed for CD11b, CD14, CD34, and CD45 markers in cattle adipose tissue-derived MSCs. The cultured MSCs can be aseptically isolated from the culture flask and injected around 107cells/animals in the site of the wound.

3.3.3 Confirmation for the presence of MSCs on wound areas of treated animals A tissue biopsy can be taken to collect the cells of wounds of animals for differentiating the injected allogeneic MSCs and their cells of animals. For this experiment, genomic DNA was isolated from biopsy cells, and PCR was performed using highly polymorphic DRB II gene primers to differentiate autologous and allogeneic cells after confirmation of the healing of the wound. Genomic DNA from the blood of allogeneic MSCs treated in animals was also analyzed in this primer to see whether allogeneic MSCs will be reached in blood or not.

FIGURE 3.2 Adipose tissues-derived mesenchymal stem cells culture of cattle. Seeding of primary ADSCs (A); ADSCs after 5 days of culture (B); Confluent ADSCs after 10 days of culture.

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FIGURE 3.3 Directed differentiation of dog adipose tissue-derived Mesenchymal stem cells into Osteocytes, Chondrocytes, Adipocytes, and Neurocytes.

3.3.4 Isolation of ovarian surface epithelium cells for generation of oocytes Ovary: The ovary sample could be collected from a slaughterhouse in 0.9% normal saline containing antibiotics (penicillin 100 IU/mL, streptomycin 100 mg/mL) at ambient temperature. Culture of Ovarian Surface Epithelium: The scraped OSE cell with the medium is now transferred to a 15 mL centrifuge tube and centrifuged at 1000  g for 10 min at 25 C. The pellet was later suspended in fresh medium and cultured in DMEM/F12 supplemented with 20% fetal bovine serum, BMP-4, bFGF, LIF, and antibiotics in 5% CO2 incubator at 38.5 C for 3 weeks. The partial medium was changed every alternate day, and cultures were monitored under an inverted microscope. Cultures were terminated at the end of the 3e6 weeks period and observed the development of oocytes-like cells under an inverted microscope.

3.3.5 Characterization of OSE-derived primordial germ cell-like structure Germ cell markers for Immunostaining - VASA, DAZL, STELLA, ZP1, ZP2, ZP3, GDF9, etc. are the primordial germ cell markers expressed in different stages of development of oocytes from OSE stem cells. RT-PCR studies: Total RNA was extracted from scraped OSE cells using TRIZOL (Invitrogen) and from cells postculture for studying stem and germ cell-specific gene transcripts by semi-quantitative RT-PCR method. VASA, DAZL, STELLA, PUMI, SCP3, ZP1, ZP2, ZP3 markers were studied using RT-PCR.

3.4 Applications of embryonic and adult stem cells Most knowledge about human development has been obtained through studying model organisms, such as fruit flies, worms, frogs, mice, and animals. Human embryonic stem cell lines, which can be cultured and differentiated into a variety of cells like cardiomyocytes, neurons, pancreatic, liver, kidney, cells, etc. and tissues paralleling the earliest events in the development of the embryo, offer a unique window into human development in research work. Scientists are presently doing their research on the therapeutic potential of human embryonic stem cells, and they want to use these cells for application goal research in their laboratories. These cells have potential applications for the treatment of diabetes, cancer, heart disease, Parkinson’s, and Alzheimer’s since these chronic diseases have fewer treatment possibilities in present-day medication and prolong time to cure the patient properly.

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3.4.1 Study of diseases and how they develop Experimental animal models used to study human diseases in the laboratory do not exactly model the disease as it occurs in people. Human pluripotent stem cells, particularly patient or disease-specific lines, offer the possibility to model human disease more accurately in the laboratory. A better understanding of normal cell development will allow us to understand and correct the errors that cause disease conditions. In vitro culture of disease-causing cells is also used for disease models to control chronic diseases that are not cured in medication.

3.4.2 Stem cells: a model for screening, discovery, and development of drugs The identification of cancerous stem cells has rapidly gained attention in the field of drug discovery. The prospect of performing screens aimed at proliferation, direct differentiation, and toxicity and efficacy studies using stem cells offer a reliable platform for the drug discovery process. Advances made in the generation of induced pluripotent stem cells from normal or diseased tissue serve as a platform to perform drug screens aimed at developing cell-based therapies against conditions like Alzheimer’s, Parkinson’s disease, diabetes, and cancer. New drugs can be tested on stem cells to assess their safety before testing drugs on animal and human models. Scientists are using ESCs to differentiate into dopamine-producing neuron cells, which can be used in the treatment of Parkinson’s disease. ESCs can be differentiated to natural killer (NK) cells, which can be used for the enhancement of immunity of the body. ESCs can also be differentiated into pancreatic beta cells, which can be used as an alternative treatment for diabetes. Scientists at Harvard University were able to differentiate ESCs into insulin-producing cells and generate large quantities of pancreatic beta cells from ES. Researchers of NDRI Karnal were differentiated cardiomyocytes, and oocytes-like cells from in vitro produced embryonic stem cells in animal science for the first time in the world. This research work will help in vitro production of embryos, the study of gene regulation, and developmental biology. The ESCs can be used in vitro meat production as knockdown of myostatin genes of the muscle, which will help the faster multiplication of the muscle mass. The slaughter of animals is a cruel method to destroy the animal and imbalance the environment. In vitro meat will be a cheaper source of nonslaughter fresh meat that will be a choice of the people.

3.4.3 Transgenic animal production A transgenic animal is one that carries a foreign gene that has been deliberately inserted into its genome. Transgenic animals provide a chance to produce animals that are a source of useful human therapeutic proteins like growth hormone, insulin, human lactoferrin, etc. Transgenic animals can be easily produced by transforming stem cells, growing in vitro, with the desired gene constructed by homologous recombination. Successfully transfected cells can be used in somatic cell nuclear transfer (SCNT) to produce transgenic animals or directly inject the gene constructed into an embryo using micromanipulation. Transgenic animals can nowadays easily be produced through CRISPR/Cas9 genome editing technique. Gene is introduced from a foreign species of transgenic animals then the growth factors will be altered in the body of animals. These transgenic animals will facilitate the study of gene regulation, development of the body, and their effect on the functions of the body in everyday study. These animals can be solely designed to study the role of genes in many disease models in developmental biology. Transgenic models can be performed better for research on the disease-resistant development of medicines, the pharmacological study of the drug. Especially chronic incurable diseases like Alzheimer’s, Parkinson, diabetes, and cancer study will be perfect as a transgenic model for the study of these diseases. Many proteins produced from transgenic animals can be used as medicines, growth factors, antibodies, blood factors, nutritional, and milk supplementation, exploiting the animals as a bioreactor. Researchers are trying to produce lactoferrin, lysozyme (role in innate immunity), thrombopoietin (platelet stimulating factor), and erythropoietin (erythropoiesis), human coagulation factor IX (blood coagulation), phenylketonuria, hereditary emphysema, and manufacture of medicines to treat diseases because of their high therapeutic applications in human beings and animals. The transgenic cows produced human alpha-lactalbumin protein into their milk and purified protein to be given to babies as a better alternative to natural cow milk. Testing of vaccines is used for transgenic animals, most commonly mice and monkeys, are used for testing the safety of vaccines, and the vaccine can be used for human beings. Biosteel is a high-strength fiber produced from recombinant spider silk secreted into the milk of transgenic goats. This biosteel is 7e10 times stronger than steel, and its very high resistance to extreme temperatures range from
20 to 330 C. This biosteel has vast applications like medical products, coating of all kinds of implants, artificial ligaments, tendons, textile products, etc. This is a biological product produced from spiders and will be used for humans and animals. The

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transgenic animals used as bioreactors to produce therapeutic proteins have existed for decades; several proteins produced in these systems are now in clinical trials, and then released for approval for marketing. The ability of transgenic animals to produce complex, biologically active proteins is efficient and economically cost-effective and superior to those of bacteria, mammalian cells, transgenic plants, and insect models.

3.4.4 Therapeutic cloning The objective of this technique is to produce pluripotent stem cells that carry the nuclear genome of the patient and then induce them to differentiate into cells that may be transplanted back into the patient. The SCNT technique requires the introduction of a nucleus from an adult donor cell into an enucleated oocyte to generate a nuclear transfer (NT) embryo. By this method, autologous pluripotent stem cells are generated that have acquired the fate of stem cells. In animals, transplantation of cells derived using this technique has been successfully applied in parkinsonian mice and in humans. Therapeutic cloning may substantially improve the treatment of many incurable diseases (Alzheimer’s, Parkinson’s, diabetes, cancer) since therapy for these diseases is currently limited by the availability or immune-compatibility of tissue transplants. In this context, the emerging field of reproductive biotechnology has provided novel avenues for the manifold increase in animal production by enhancing animal productivity by using different techniques like artificial insemination, gamete preservation, in vitro fertilization, and embryo transfer. These techniques have been used for propagation of only a few populations of animals. Production of faster multiplication of a large number of low-cost genetically identical elite livestock species is still an attractive goal for the researchers of the animal sector. Somatic cell nuclear transfer (SCNT) is one of the best techniques for faster multiplication of livestock in biotechnological tools. Nuclear cloning of elite animals with a proven genetic background is generally used to yield superior animals for the faster multiplication of livestock species. This cloning technique is a combination of classical reproduction, cellular, and molecular biological and genomic techniques for enhancing the productivity of the animal. Cloned embryos can be used for embryonic stem cell production, which can be used for the treatment of diseases of animals.

3.4.5 Regenerative medicine The term “regenerative medicine” is often used to describe medical treatments and research that use stem cells, either adult or embryonic, to restore the function of organs or tissues. This can be achieved either by administering stem cells or specific cells that are derived from stem cells in the laboratory or by administering drugs that stimulate stem cells that are already present in tissues to more efficiently repair the tissue involved. In theory, any condition in which there is tissue degeneration can be a potential candidate for stem cell therapies, including mastitis, metritis, Parkinson’s disease, spinal cord injury, heart disease, Type 1 diabetes, muscular dystrophies, retinal degeneration, and liver disease.

3.5 Current clinical applications of adult mesenchymal stem cells in regenerative medicine Mesenchymal Stem Cells (MSCs), a kind of adult stem cell, are extensively used as regenerative medicine. The main clinical application potentials of MSCs involve the transplantation of autologous or allogeneic cells into patients, through a local or systemic infusion. The stem cell transplantation can be used for a broad spectrum of indications, including cardiovascular disease, lung fibrosis, spinal cord injury, and bone and cartilage repair. The first clinical trial using in vitroderived MSCs was carried out in patients who became the recipients of the autologous cells. Since then, a revolution came toward the use of MSCs for the treatment of the number of diseases, and many clinical trials have been conducted to test the feasibility and efficacy of MSCs therapy. The public clinical trials database showed that many clinical trials have been carried out using MSCs for a very wide range of therapeutic applications (Trounson and McDonald, 2015; Lukomska et al., 2019). Isolated from bone marrow, adipose tissue, umbilical cord, blood, nerve tissue, and dermis, MSCs can be administered both systemically and locally for the treatment of different wounds. MSCs have been shown to express low levels of longterm incorporation into healing wound areas for the release of trophic mediators, rather than a direct structural contribution. They generally release the vascular endothelial growth factor (VEGF), stromal cell-derived factor-1, epidermal growth factor, keratinocyte growth factor, insulin-like growth factor, and matrix metalloproteinase-9. They promote new vessel formation, recruit endogenous progenitor cells, and direct cell differentiation, proliferation, and extracellular matrix formation during wound repair (Duschera et al., 2016). According to the International Society of Cellular Therapy (ISCT),

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Mesenchymal stem cells adhere to a plastic surface, and they express their surface markers CD73, CD90, and CD105, and they are unable to express their hematopoietic markers CD14, CD34, CD45, CD11b/CD79, and CD19/HLA-DR. MSCs can differentiate into osteoblastic, adipocytic, neurocyte, and chondrocyte lineages. MSCs also show key immunomodulatory properties through the secretion of interferon-l, interleukin-1a, tumor necrosis factor-a interleukin-1b, and nitric oxide synthase. MSCs secrete prostaglandin E2 and regulate fibrosis, as well as inflammation, promoting tissue healing. Not only that, MSCs also have bactericidal properties through the secretion of antimicrobial factors and by upregulating bacterial killing and phagocytosis by immune cells. Mesenchymal stem cells (MSCs), the major stem cells for cell therapy, have been used in the treatment of animals. From animal models to clinical trials, MSCs have afforded promise in the treatment of many diseases, primarily, tissue injury and immune disorders. However, MSCs for cell therapy is safe and effective (Wei et al., 2013). Cytokines are small proteins that are important in cell signaling. It is produced by cells like macrophages, T lymphocytes, B lymphocytes, and mast cells, fibroblasts, endothelial cells, and various stromal cells. Cytokines that are secreted in MSCs derived from adipose tissue for cell signaling proteins are Leptin, Adiponectin, plasminogen activator inhibitor-1 (PAI-1), chemerin, interleukin-6 (IL-6), Apelin, monocyte chemotactic protein-1 (MCP1), retinol-binding protein-4 (RBP4), visfatin, omentin, vaspin, progranulin tumor, necrosis factor-alpha (TNFa), etc. These are also involved in autocrine signaling, paracrine signaling and endocrine signaling as immunomodulating agents. Cytokines are regulators of host responses to immune responses, infection, inflammation, and trauma in the muscles of the body. Other reports have clearly shown that MSCs secrete immunomodulatory, proangiogenic, promitogenic, antiapoptotic, antiinflammatory, antibacterial factors, and antivirus-like transforming growth factor b-1, interleukin-10, hepatocyte growth factor, heme oxygenase-1, prostaglandin E2, and HLA-G5. MSCs are also secreted by trophic factors like brain-derived neurotrophic factor (BDNF) in response to autocrine interferon (IFN)-b, glial cell line-derived neurotrophic factor (GDNF), nerve growth factor (NGF), angiopoietin (ANG), and angiogenic vascular endothelial growth factor (VEGF). These stem cells are used to develop stem cell-based therapies for many diseases like orthopedic, cardiovascular, ophthalmic, and neurological disorders. ES cells are developed in teratoma when injected into the body, but MSCs themselves do not form any tumor-like growth. Therefore, any immunosuppressive drugs are not required for autologous and allogeneic MSCs treatment as MSCs have the immunomodulatory function, leading to preventing GVHD. MSCs have also inhibited the proliferation of Th1, CD4(þ)T cells, CD8(þ) T cells, Th17 cells, and direct inhibition of natural killer cells. Donor CD4(þ) T cell proliferation and human tumor necrosis factor (TNF) reduction in serum. MSCs are mostly immune-evasive cells as they do not express MHC class II antigens and costimulatory molecules like CD40, CD80, and CD86, and minimally express MHC class I antigens in the cells. Due to the lack of immunogenicity, MSCs facilitate clinical therapy, including for allogeneic cell therapy in patients, as it shows very low immunogenicity and high immunosuppressive potential. It has been reported that MSCs do not suffer from immune rejection and the least ethical issues as these cells are generally used as autologous and allogeneic cells for stem cell transplantation. Studies have shown that even at high doses, treatment with adipose-derived MSCs caused no serious side effects. In addition, no immune-rejection responses or tumor developments have been observed. Antibiotic-resistant bacterial diseases are increasing and need more effective treatment, including alternatives to conventional antibiotics or stem cell therapy. Staphylococcus aureus causes mastitis and metritis with high mortality and many chronic implant diseases worldwide. Nowadays, MSC has attracted attention to control bacterial infections based on in vitro studies documenting direct bactericidal activity to direct administration of MSC controls bacterial diseases to overcome conventional antibiotic treatment. It is reported that MSCs exert strong antimicrobial effects through direct and indirect mechanisms, partially mediated by the expression of antimicrobial proteins and peptides (Miranda et al., 2017). MSCs have also been shown to improve wound healing and suppress inflammatory immune responses as MSC exhibits antimicrobial activity. It has been reported that in vitro and in vivo studies can rest and activate MSC to kill bacteria, including multidrug-resistant strains. MSC generates multiple direct and indirect, immunologically mediated antimicrobial activities that combine to help eliminate chronic bacterial infections when the cells are administered therapeutically. For defining human MSCs are (a) adherence to the plastic surface, (b) specific surface antigen expression (Positive expression of CD105, CD73, and CD90, and lack of expression of CD45, CD34, CD14 or CD11b, CD79a or CD19, and HLA-DR), and (c) multipotent differentiation potential for osteoblasts, adipocytes, and chondroblasts using standard in vitro tissue culture-differentiating conditions (Dominici et al., 2006). The presence of MSCs in normal skin and their critical role in wound healing suggest that the application of exogenous MSCs is a promising solution to treat nonhealing wounds. MSCs have a role in the inflammatory, proliferative, and remodeling phases of wound healing, and their presence supports healthy physiologic functioning toward successful healing. As such, the therapeutic application of MSCs has been shown to enhance and improve wound healing in clinical settings (Maxson et al., 2012).

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Intravenous injection of MSCs showed bacterial clearance was significantly greater in MSC-treated mice due to increasing phagocytotic activity of the immune cells of the host. It has been reported that intravenous injection of MSCs improves myocardial infarction in mice as intravenous is the least invasive route, and the cells are infused into the venous blood supply, and the cells will migrate faster toward the injured tissue sites. The cells are generally engrafted in the lungs and other major target organs and move very rapidly to the site of injury, as these cells have been detected already seconds or minutes after intravenous transplantation (Lee et al., 2009). It is, therefore, for better therapy, systemic administration will be preferable compared to topical administration when stem cells are used for controlling antiinflammation and immunosuppression effects. Although intravenous delivery is a simple route of administration for the therapy, this method can possibly produce pulmonary embolism, though this has not been recorded in humans. Intraperitoneal injection (IP), Intravenous injection (IV), or anal injection (AI) is the best way for MSCs transplantation for many diseases.

3.5.1 Treatment of massive wounds of animals Lameness is the third most expensive dairy disease, following mastitis and reproductive failure (Juarez et al., 2003). The problems in the hoof lead to lameness in the animals rendering them useless. Many studies have demonstrated that lameness reduces fertility performance like prolonged interval from calving to the first service, and lower reproductive efficiency (Somers et al., 2015) in the dairy cows. Lameness has a very high incidence, even in well-managed farms. The prevalence of lameness in the USA dairy herd in free-stall housing averages around 25% (Liang, 2013). The lameness costs include treatment costs, labor, discarded milk, reduced milk yield, increased culling risk, extended CI, veterinarian service fees, and extra services. The total costs were an average of $421.53 per case. In India at NDRIKarnal, (Singh et al., 2014) compared the production performance of 96 lame cows with 67 healthy Karan Fries crossbred cows. A significant total loss of 498.95 kg of milk was observed during 305 days. Effect of lameness hoof disorders on the productivity of Karan Fries crossbred cows. 2011, Animal Science Journal). Highly contagious livestock disease FMD causes hoof wounds in animals, leading to lameness for prolonging time and becoming unproductive. The crossbred cattle and indigenous buffaloes are also suffering from hoof wounds and becoming unproductive. Stem cells escalate the wound healing in clinical, as well as preclinical wounded animals. MSCs, help in wound healing by releasing antiinflammatory cytokines and various tissue repair growth factors. The MSCs are injected aseptically near the wound of the animal. Autologous, as well as paralogous injections of stem cells, can be administered in the wounded animal. Massive wounds in dogs were healed by the administration of allogeneic stem cells (Fig. 3.4) (Malik et al., 2013). Similarly, Chronic hoof wounds of cattle and buffalo were successfully treated by using in vitro cultured MSCs. The authors also observed the regeneration of new skin and hairs around the healing wound in all the animals (Fig. 3.5) (Chuong, 2007, Malakar et al., 2019).

3.5.2 Mastitis treatment In mastitis, the udder tissue of animals gets damaged to the extent that milk production by the animal ceases. By using stem cells, the mammary gland epithelial cells have been generated by the scientists successfully, which gives a ray of hope for the treatment of mastitis. Due to mastitis, the animals are inducing a huge economic loss in the world leading farmers to a grave condition. Mastitis may be the most important disease in dairy cattle and buffaloes on account of huge economic losses worldwide. The annual economic loss due to mastitis has been calculated to be Rs. 7165.51 crores in India. Total

FIGURE 3.4 Treatment massive wound of a dog with Mesenchymal stem cell: The Ulna bone was showing (A). Growth of muscle cells after 7 days in the wound area (B). The wound was healed, and the skin is also growing after the treatment of mesenchymal stem cells after 14 days (C).

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FIGURE 3.5 Treatment hoof wound of a cow with Mesenchymal stem cell and completely cured within 40 days.

economic loss due to subclinical mastitis has been estimated at 4151.16 crores (PDADMAS, 2011). After FMD, mastitis may be the second most important disease in dairy cattle and buffaloes in India on account of huge economic losses. Studies in the USA show that costs related to mastitis are approximately U$ 200 per cow/year. This gives an annual loss of two billion dollars to the dairy industry. According to another study, the estimated loss due to mastitis in cows was almost 700 kg in the first lactation and 1200 kg in the second or higher lactation. Mastitis can be treated with MSCs and all the animals are cured by secreting normal milk. Sporadic use of antibiotics is becoming antibiotic resistance to animals, and the residues are also coming through milk leading to human health hazards. We have treated the mastitis cows with MSCs, and all the cows were cured and assessed the efficiency of MSCs for the treatment of mastitis cattle (Fig. 3.6). First, the identification and classification of animals were performed as per the veterinary clinical records. Mastitis cases were identified based on SSC and CMT scores. Farmers in our country are unnecessarily spending huge amounts of money to treat their animals, but MScs are cheaper and easily cure mastitis (Malakar et al., 2019).

3.5.3 Metritis and endometritis Metritis is an inflammation of the uterine lining due to infection that is commonly initiated at parturition. Endometritis has an economic impact as it reduces fertility and milk yield and is associated with an increase in culling rates. It is reported that between 10% and 15% of dairy herds are suffering from endometritis; however, it is variable from herd to herd, with a total cost of £160 per case. Metritis cases were identified based on polymorphonuclear cells (PMN) counts of vaginal secretion. Treatment of mastitis and metritis was performed with allogeneic mesenchymal stem cells @ dose 107e108

FIGURE 3.6 Treatment of mastitis of a cow with mesenchymal stem cells and cured within 28 days.

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cells/injection at the route of delivery. Local and Intravenous injections at an interval of 15 days. All the metritis suffering cattle are cured with the treatment of MSCs. We have determined the efficacy of MSCs in treating mastitis and metritis in the bovine by intravenous application of MSCs. Clinical improvement was assessed by signs and symptoms of healing on every progressive day. Immunomodulatory molecular profile was also performed with real-time PCR as blood cells were collected weekly to see the secretion of antiinflammatory cytokine: IL10, TNF alpha, angiogenic vascular endothelial growth factor (VEGF) and angiopoietin (ANG) and antimicrobial cytokine: IL37.

3.5.4 Bone fracture and orthopedic defects Although bone has natural self-healing properties, the properties of bone change throughout the life of an animal. Aging severely affects the healing potential of bones. Thus, there is a need for alternative therapies which ensure fast bone regeneration. The therapeutic potential of MSCs in healing bones has already been examined extensively. MSCs may act by directly differentiating into bone cells, attracting and homing other cells, or creating a regenerative environment by producing trophic growth factors (Lin et al., 2019). There are several studies worldwide in which MSCs were used to heal the fractured bones (Hu et al., 2018, Malakar et al., 2019). In 2011, patients suffering from osteonecrosis of the femoral head and knee osteoarthritis were treated by a combination of percutaneously injected autologous adipose-derived stem cells, hyaluronic acid, platelet-rich plasma, and calcium chloride (Pak, 2011). At 3 months, in all cases, pain and mobilization were improved and significant filling of bone defects.

3.5.5 Spinal cord injury Spinal cord injury is among the most intricate and diverse pathological disorders of CNS (central nervous system) impairments. Spinal surgery becomes challenging due to the involvement of both orthopedic and neurosurgery. Owing to the heterogeneous differentiation of MSCs and risks of complex spinal surgeries, stem cell regenerative therapy is emerging as a subject of interest for the treatment of the spinal injury. It has been demonstrated that MSCs can produce several growth factors, including chemokines and neuroprotective cytokines, which help in faster healing of the injured spinal cord after aseptic transplantation (Novikova et al., 2011; Awad et al., 2015). In 2011, patients suffering from spinal cord injury were treated with intravenous infusions of autologous adipose-derived stem cells. For 12 weeks, motor function was improved in patients (Arboleda et al., 2011).

3.5.6 Treatment of dogs Stray dogs and pet dogs suffer from a variety of injuries such as massive wounds due to dog bites or strangulation at the neck region by rope, fractures, muscular dystrophy, hip dysplasia, burn wounds, diabetes, kidney problems, etc. MSCs transplantation provides a safe and relatively easy method to restore these injuries. Xenogeneic MSCs derived from humans were injected systemically into the cephalic vein of the golden retriever dog suffering from Duchenne muscular dystrophy (DMD), which resulted in the fully recovered dog with no side effect (Pelatti et al., 2016). In another study, one dog suffering from hip dysplasia and another from paraplegia were cured by transplantation of allogeneic adipose-derived mesenchymal stem cells in 1 month of time (Malik et al., 2016). Dogs are suffering from paralysis, wound, spinal cord injury and the animal models facing unimaginable problems can be treated with mesenchymal stem cells.

3.5.7 Blood stem cell transplantation Bone marrow contains blood-forming stem cells (hematopoietic stem cells) that have been used for decades to treat blood cancers (leukemia, lymphoma) and other inherited blood disorders (sickle cell anemia or some metabolic conditions). Umbilical cord blood is another source of hematopoietic stem cells that are being used in the treatment of different diseases. Doctors have been transferring blood stem cells to bone marrow transplants for more than 40 years.

3.5.8 Burn therapy Healing of burn wounds involves a sequence of intricate processes that are dependent on the quality of the wound and the healing time. Severe burn injuries have stressful effects on the affected animal leading to decreased productivity. MSCs therapies have been recently applied in the field to enhance significant wound healing (Fig. 3.7). It has been shown that stem cells promote improved and faster healing of burn wounds (Koenen et al., 2015). Furthermore, stem cells help in reducing the inflammation levels with less wound progression and fibrosis (Wu et al., 2014).

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FIGURE 3.7 Mesenchymal stem cells were injected into the burn areas of a heifer, and the skin and hair were grown in the burn areas.

3.5.9 Corneal regeneration Limbal stem cells have been used successfully to restore vision in patients suffering from chemical destruction of the cornea (Kolli et al., 2010). Clinical trials are on for hESC-derived retinal cells to treat patients with an eye disease called Stargardt’s Macular Dystrophy (SMD) and age-related macular degeneration. A group of scientists in 2012 used embryonic stem cells to restore partial vision in patients who were legally blind (Pearson et al., 2012).

3.5.10 Immunomodulatory disease treatments The hypoimmunogenic nature of MSCs shows that they have broad implications in terms of allogeneic therapy, or the delivery to a recipient of cells derived from an unmatched donor. There are several reports describing the clinical use of allogeneic donor-mismatched cells with little evidence of host immune rejection or Graft-versus-host-disease (GVHD). For example, allogeneic bone marrow transplantation in children with Osteogenesis Imperfecta resulted in engraftment of donor-derived MSCs and an increase in new bone formation. In 2006, transplantation of MSCs to eight patients with steroid-refractory grades III-IV GVHD and one who had extensive chronic GVHD showed that acute GVHD disappeared completely in six of eight patients (Ringdén et al., 2006).

3.5.11 Neurodegenerative diseases The nervous system in our body is a complex and large tissue that controls many important functions like smell, sight, feel, sound, taste, voluntary, and involuntary muscle movements that are autonomous or as per our ability to reason. In the body, many types of neurons are correctly wired and supported by glial and other supportive cells that coordinate all tissue function. Degeneration of these neuron cells causes many neurodegenerative diseases like Alzheimer’s, Parkinson’s, and Huntington’s diseases, and there is no curative medicine presently available. Stem cell therapy is the only therapy to restore the function of the ailment of the nervous system, which manufactures neural cells and can be surgically transplanted into damaged areas for restoration of the function of the neuron. There are many approaches to restore brain function through cell transplantation therapies like the proliferation of resident cells, fetal or adult stem and progenitor cells, pluripotent stem cells, and IPS cells. Presently research on the transdifferentiation of nonneuronal cells like MSCs into neuronal tissues is the main target to restore the function of the neuron. Clinical trials have involved grafting brain tissue from aborted fetuses into patients with Parkinson’s, Alzheimer’s, and Huntington’s disease. While some successes have been noted after transplantation of the MSCs into the damaged areas and the results have not been uniformed in these clinical trials, further clinical trials will involve more refined patient selection, in an attempt to improve the restoration of the proper function of the neuron cells of the brain. All the patients will be benefited from stem cell treatment therapy as these diseases are chronic, incurable, and longer time sufferers of the patients (Volkman and Offen, 2017).

3.5.12 Liver diseases Acute or chronic liver failure occurs in cirrhotic patients of nearly 30% who are hospitalized. But the liver transplant is only available only to 10% of patients who spend a huge amount of money each year. Morbidity and mortality rates of the cirrhotic patients are always remaining high in most of the countries. MSCs are used for the treatment of cirrhotic patients who are gradually cured. A clinical trial was carried out by autologous injection of MSCs, on eight patients (four hepatitis B, one hepatitis C, one alcoholic, and two cryptogenic) with end-stage liver disease. All patients tolerated well, and their liver function improved, suggesting the feasibility, safety, and efficacy of using MSCs as a treatment for end-stage liver disease.

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3.5.13 Cardiac related diseases MSCs can differentiate not only into osteoblasts, neurons, chondrocytes, vascular endothelial cells, skeletal muscle cells but also into cardiomyocytes. MSCs can be directly differentiated into beating cardiomyocytes using the 5-azacytidine treatment in in vitro conditions. It was reported that these MSCs can be directly injected into an infarcted heart that has been shown to induce myocardial regeneration and improve cardiac function. Implantation of MSCs is induced by therapeutic angiogenesis in a rat model of hindlimb ischemia through vascular endothelial growth factor (VEGF) production. It has also been reported that myocardial blood flow abnormalities have been cured with MSCs. MSCs derived from bone marrow when delivered locally, can generate de novo myocardium, showing that stem cell therapy can be used to treat coronary artery disease (Trounson et al., 2011). The practical utility of this approach was demonstrated in patients suffering from myocardial infarction, by the delivery of bone marrow-derived MSCs and the patients showed a dramatic improvement in their global heart function. MSC transplantation in a rat model of dilated cardiomyopathy resulted in increased capillary density and decreased collagen volume fraction in the myocardium. Transplantation of MSCs is induced by myogenesis, angiogenesis, and decreases collagen deposition in the myocardium, and improves cardiac function in a rat model. MSCs are also differentiated into cardiomyocytes and vascular cells, which are providing angiogenic, antiapoptotic, and mitogenic factors. This transplantation is mediated by the downregulation of proinflammatory cytokines like interleukin (IL)-6 and tumor necrosis factor (TNF) and upregulation of antiinflammatory cytokines like IL-10 and IL-4.

3.5.14 Treatment of diabetes with MSCs Diabetes are more prevalent in human beings, and animals suffer for a prolonged time. Diabetes is expected to increase by 438 million by 2030 due to a lack of insulin. WHO estimates that 80% of diabetic deaths occur, and projected deaths will be double up to 2030. India is the largest diabetes capital in the world and suffers 62 million people. Nearly 177 million people worldwide are diabetic, and it will be double by 2030. MSCs are a new herald for the treatment of incurable diseases like mastitis, wounds, fracture, diabetes, and cancer. These cells are self-renewing multipotent cells that have the capacity to secrete multiple biologic factors that can restore and repair injured tissues in the body. It is evidenced that preclinical and clinical pieces of evidence have the therapeutic benefit of MSCs in various medical treatments. MSCs are generally used in cell-based therapy in the control of diseases because of their regenerative potentiality, ease of isolation, and low immunogenicity. Experimental and clinical studies have provided promising results for MSCs for the treatment of diabetics patients. In 2010, the results obtained on allogeneic injection of adipose tissuederived stem cells in diabetic patients resulted in improved health, the gain of weight, and the gradual decrease in insulin requirements in patients.

3.5.15 Treatment of cancer with MSCs Cancer burden rises to 18.1 million new cases and 9.6 million cancer deaths in 2018. Cancer affects 90.5 million people every year in the world (WHO, 2018). Cancer cases of 17.35 lakh and 6.09 lakh deaths are projected in the United States in 2018 (Siegel et al., 2020). Total deaths due to cancer are 8.8 million, or 15.7% of deaths (GBD, 2017). The financial loss due to cancer is $1.16 trillion per year (WCR, 2014). The total medical cost for cancer in the USA is $80.2 billion in 2015. Every year thousands of women are diagnosed with breast cancer globally (Tew et al., 2014) and rising trends. Similarly, in India, 14.5 lakh people are suffering from cancer, and about seven lakh new cases are registered every year. Presently there is no effective permanent treatment available for these diseases in India. MSCs are a new herald for the treatment of cancer disease. MSCs treatment shows profound therapeutic potential for treating various human and animal diseases, including cancer. MSCs are considered the best promising source of stem cells in autologous cell-based therapies. These cells can be used to target cancer cells as it has inherent tumor-tropic properties. MSCs can also regulate the growth of cancer cells through autocrine and paracrine mechanisms and control the growth of cancer cells.

3.6 Challenges of stem cells 3.6.1 Stem cells in reproduction and infertility Spermatogonial stem cells (SSCs) are a type of adult stem cells found in male mammals. These cells have the capacity for self-renewal and are capable of differentiating in the niche of the testis. They are also the only adult stem cells in a normal postnatal body that undergo self-renewal throughout life, transferring genetic information to the offspring. These cells can be exploited in three ways:

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3.6.2 Testis xenografting The primary clinical application for testis xenografting would be as a means to preserve the breeding potential of a genetically valuable prepubertal male animal. For example, in the captive management of threatened or endangered species, specific individuals often have high genetic value. If neonatal or juvenile males die, testis xenografting offers a means to develop sperm from their gonocytes or SSCs, which are present in parturition. In this procedure, small pieces of donor testes are surgically grafted into immunodeficient mice. In the absence of a functioning immune system, the recipient mice nurture the foreign testis tissue, which supports spermatogenesis.

3.6.3 Spermatogonial stem cell transplantation The primary clinical use of SSCT would be to preserve or manipulate the male germline of both. If exogenous DNA is integrated into the chromosomes of SSCs, the sperm differentiated from these SSCs are likely to carry the foreign gene; therefore, chances of transfer of foreign genes to offspring increase. This technology provides an excellent approach to deliver exogenous gene-carrying SSCs into the embryonic genome, and thus, has a significant impact on the production of transgenic animals. Potential benefits of the Spermatogonial stem cell transplantation technique to livestock production include enhanced dissemination of elite animal genetics as this would permit a continuous supply of transplantation into less-valuable recipient animals, cross-breeding in harsh environments, conservation of endangered species, production of transgenic animals, use of progeny-tested animals in diseased conditions and even after death, conversion of the genetically inferior male into a genetically superior male.

3.6.4 Spermatogonial stem cells as a source for fertility restoration Spermatogonial stem cell loss is an important cause of male infertility. Stem cell loss can occur after chemotherapy and radiotherapy or due to a genetic disease. Because children do not have the possibility to bank spermatozoa, the preservation and transplantation of SSCs may become an important strategy to treat reproductive stem cell loss disorders. Banking and transplantation of SSCs may become a promising method to preserve the fertility of prepubertal patients.

3.6.5 Generation of oocytes from ovarian surface epithelium for regenerative medicine According to the earlier belief, in the human embryo, thousands of oogonia divide rapidly from the second to the seventh month of gestation to form around seven million germ cells/oocytes. After the seventh month of embryonic development, the number of germ cells gradually drops precipitously. Most oogonia die during this period, while the remaining oogonia enter the first meiotic division. These latter cells, called the primary oocytes, progress through the first meiotic prophase until the diplotene stage of cell division. At this point, they are maintained until puberty. With the onset of adolescence, groups of oocytes gradually and periodically resume meiosis. Thus, in the human female, the first part of meiosis begins in the embryo, and the signal to resume meiosis is not given until around 12 years later. Some oocytes are maintained in meiotic prophase for nearly 50 years. Primary oocytes continue to die even after birth. Of the millions of primary oocytes present at birth, only about 400 maturing during a woman’s lifetime can be observed. In vitro production of oocytes from the ovarian surfaces, epithelial stem cells will lead to the generation of a large number of oocytes from a single ovary (Fig. 3. 8) (Fatima et al., 2015). This technique will have an immense application to faster multiplication of elite animals, solution of infertility of animal and human being, transgenesis, and conservation of livestock and endangered species (Singhal et al., 2015).

FIGURE 3.8 Differentiation of ovarian surface epithelial stem cells into oocytes. (A) and (B) oocytes like cells were observed on the culture at the end of 4 weeks.

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3.7 Conclusion In the present day, adult mesenchymal stem cell therapy could be possible to cure mastitis, metritis, and hoof wounds of cattle and buffaloes permanently within a short period of time. As the available reports, these diseases cause huge economic loss in our country. We have already treated mastitis, metritis, and hoof wounds of cattle and buffaloes with mesenchymal stem cells and cured all the animals completely and permanently. Treatment of MSCs can drastically reduce the present treatment cost on antibiotics and other therapies to the suffering animals. The farmers will be highly benefited as their animals will be given more milk, increase their conception rate, improve the health of animals, and remove their agony due to their suffering animals. MSCs can also be used for the treatment of different diseases like diabetes, cancer, cirrhosis, cardiac diseases, etc. Germ cells can be generated from embryonic stem cells, iPS cells, Spermatogonial stem cells. These stem cells are promising cells for regenerative medicine and translational sciences for the treatment of many diseases in humans and animals.

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Malik, H.N., Dubey, A., Singhal, D.K., Saugandhika, S., Boeteng, S., Fatima, S., Singhal, R., Sharma, V., Saini, S., Kumar, S., Guha, S.K., Malakar, D., 2013. Isolation, characterization, and differentiation of adipose tissue-derived mesenchymal stem cells: autologous transplantation to patients. Reprod. Fertil. Dev. 26 (1), 216. Maxson, S., Lopez, E.A., Yoo, D., Miagkova, A.D., LeRoux, M.A., 2012. Concise review: role of mesenchymal stem cells in wound repair. Stem Cells Transl. Med. 1 (2), 142e149. Miranda, F.A., Cuenca, J., Khoury, M., 2017. Antimicrobial activity of mesenchymal stem cells: current status and new perspectives of antimicrobial peptide-based therapies. Front. Immunol. 8, 339. Novikova, L.N., Brohlin, M., Kingham, P.J., Novikov, L.N., Wiberg, M., 2011. Neuroprotective and growth-promoting effects of bone marrow stromal cells after cervical spinal cord injury in adult rats. Cytotherapy 13 (7), 873e887. Okita, K., Ichisaka, T., Yamanaka, S., 2007. Generation of germline-competent induced pluripotent stem cells. Nature 448 (7151), 313. Pak, J., 2011. Regeneration of human bones in hip osteonecrosis and human cartilage in knee osteoarthritis with autologous adipose-tissue-derived stem cells: a case series. J. Med. Case Rep. 5 (1), 296. PDADMAS, 2011. PDADMAS news. Project Directorate on Animal Disease Monitoring and Surveillance, vol. 1. PDADMAS, p. 8, 1. Pearson, R.A., Barber, A.C., Rizzi, M., Hippert, C., Xue, T., West, E.L., Duran, Y., Smith, A.J., Chuang, J.Z., Azam, S.A., Luhmann, U.F.O., 2012. Restoration of vision after transplantation of photoreceptors. Nature 485 (7396), 99. Pelatti, M.V., Gomes, J.P.A., Vieira, N.M.S., et al., 2016. Transplantation of human adipose mesenchymal stem cells in non-immunosuppressed GRMD dogs is a safe procedure. Stem Cell Rev. & Rep. 12 (4), 448e453. Ringdén, O., Uzunel, M., Rasmusson, I., et al., 2006. Mesenchymal stem cells for the treatment of therapy-resistant graft-versus-host disease. Transplantation 81 (10), 1390e1397. Siegel, R.L., Miller, K.D., Jemal, A., 2020. Cancer statistics, 2020. CA: Cancer J. Clin. 70 (1), 7e30. https://doi.org/10.3322/caac.21590. Singh, D., Kumar, S., Singh, B., Bardhan, D., 2014. Economic losses due to important diseases of bovines in central India. Vet. World 7, 6. Singhal, D.K., Singhal, R., Malik, H.N., Singh, S., Kumar, S., Mohanty, A.K., Kaushik, J.K., Malakar, D., 2015. Generation of germ cell-like cells and oocyte-like cells from goat induced pluripotent stem cells. J. Stem Cell Res. Ther. 5, 279. Somers, J., Huxley, J., Lorenz, I., et al., 2015. The effect of Lameness before and during the breeding season on fertility in 10 pasture-based Irish dairy herds. Irish. Vet. J. 68 (14) https://doi.org/10.1186/s13620-015-0043-4. Takahashi, K., Yamanaka, S., 2006. Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell 126 (4), 663e676. Tew, W.P., Muss, H.B., Kimmick, G.G., Von Gruenigen, V.E., Lichtman, S.M., 2014. Breast and ovarian cancer in the older woman. J. Clin. Oncol. 32, 2553e2561. Thomson, J.A., Itskovitz-Eldor, J., Shapiro, S.S., et al., 1998. Embryonic stem cell lines derived from human blastocysts. Sci. Technol. Humanit. (5391), 1145e1147. Trounson, A., McDonald, C., 2015. Stem cell therapies in clinical trials: progress and challenges. Cell Stem Cell 17 (1), 11e22. Trounson, A., Thakar, R.G., Lomax, G., Gibbons, D., 2011. Clinical trials for stem cell therapies. BMC Med. 9 (1), 52. Volkman, R., Offen, D., 2017. Concise review: mesenchymal stem cells in neurodegenerative diseases. Stem Cell. 35 (8), 1867e1880. WCR (World Cancer Report), 2014. Global battle against cancer won’t be won with treatment aloneeEffective prevention measures urgently needed to prevent cancer crisis. Press release N 224 London. Wei, X., Yang, X., Han, Z., Qu, F., Shao, L., Shi, Y., 2013. Mesenchymal stem cells: a new trend for cell therapy. Acta Pharmacol. Sin 34, 747e754. WHO (World Health Organization), 2018. The International Agency for Research on Cancer (IARC), Latest global cancer data: Cancer burden rises to 18.1 million new cases and 9.6 million cancer deaths in 2018. Press release N 263, at Geneva, Switzerland. Wu, Y., Huang, S., Enhe, J., Ma, K., Yang, S., Sun, T., Fu, X., 2014. Bone marrow derived mesenchymal stem cell attenuates skin fibrosis development in mice. Int. Wound J. 11 (6), 701e710.

Chapter 4

Alternative transcriptome analysis to build the genome-phenome bridges in animals Xuelei Han, Songlei Xue, Amy L. Zinski, Shane Carrion, Jennifer J. Michal and Zhihua Jiang Department of Animal Sciences, Washington State University, Pullman, WA, United States

4.1 Introduction Genome-wide use of alternative sites for transcription start, termination, and splicing contribute significantly to transcriptome diversity, dynamics, and flexibility, which are all essential for gene regulation in animals (Shi, 2012). In principle, the expression of a large number of alternative transcripts enables a finite genome to build an infinite phenome because almost every gene will execute quantitative, qualitative and epigenetic functions during a lifespan. Alternative transcripts are specific to cell growth, development, and differentiation and are uniquely expressed according to physical, physiological, pathological, and even psychological challenges (Elkon et al., 2013; Shi, 2012; Tian and Manley, 2013). As such, accurate profiling of alternative transcription for every gene will aid in the discovery of causal mutations that underlie health and diseases, selection of regulatory switches for functional characterization and formation of biomarker panels for disease diagnosis and drug discovery (Diederichs et al., 2016; Le et al., 2015; Otonkoski, 2016). In fact, alternative transcription is largely due to the use of alternative transcription start (ATS) and polyadenylation (APA) sites, rather than by use of alternative splicing sites(Pal et al., 2011; Reyes and Huber, 2018). The current estimate is that the human genome uses w200,000 ATS and w450,000 APA sites (Harrison et al., 2019; Lizio et al., 2019). Therefore, this chapter will mainly focus on advances in alternative transcriptome analysis, including ATS and APA profiling, and their application in functional annotation of animal genomes.

4.2 Modern sequencing platforms and transcriptome profiling strategies 4.2.1 High-throughput sequencing technologies Over the last 15 years, high-throughput sequencing platforms have advanced a lot, significantly revolutionizing transcriptome profiling (Jiang et al., 2015). These technologies are classified into either next-generation or third-generation sequencing (NGS and TGS) methods (Table 4.1) (Besser et al., 2018; Cao et al., 2017; da Fonseca et al., 2016; Hodkinson and Grice, 2015; van Dijk et al., 2018). The Roche 454, the Applied Biosystems SOLiD (supported oligonucleotide ligation and detection), the Ion Proton, and the Solexa Genome Analyzer platforms are examples of the NGS platforms. The first three NGS methods are now owned by Life Technologies (Grand Island, NY), while the last one was developed by Illumina (San Diego, CA). The PacBio RSII/Sequel systems created by Pacific Biosciences (Menlo Park, CA) and the MinION/GridION/PromethION systems made by Nanopore (Oxford, England) are the examples of the TGS technologies as they produce long reads in comparison to the short reads generated by the NGS platforms. Among these six highthroughput sequencing platforms, only SOLiD uses sequencing by ligation; the remaining systems carry out sequencing by synthesis. All three Life Technologies platforms use emulsion PCR, whereas the Illumina platform carries out bridge PCR to prepare the “clonal amplification” of templates for sequencing. In comparison, the PacBio and Nanopore platforms use single molecules as templates for sequencing. The preparation of low-diversity libraries must be avoided to ensure

Advances in Animal Genomics. https://doi.org/10.1016/B978-0-12-820595-2.00004-7 Copyright © 2021 Elsevier Inc. All rights reserved.

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50

Company

Platform

Read length

Yield/run/ Flow cell

Features

Life Technologies

454 GS 20

100 bp

60 Mb

1,4,6,12,14

454 GS FLX

250 bp

150 Mb

2. Sequencing by ligation

454 FLX titanium

400 bp

500 Mb

3. Sequencing by single-molecule synthesis

454 FLX titanium XLþ

700 bp

700 Mb

4. Emulsion PCR

SOLiD

50e75 bp

300 Gb

2,4,8,13

5. Bridge PCR

Ion torrent PGM

Up to 400 bp

0.08e2 Gb

1,4,7,12,16

6. Pyrophosphate detection

Ion torrent S5

Up to 400 bp

0.6e15 Gb

7. Proton detection

Ion torrent proton

Up to 200 bp

10e15 Gb

8. Fluorescence: di-base probes

MinSeq

1  75e2  150 bp

1.7e7.5 Gb

MiSeq

1  36e2  300 bp

0.3e15 Gb

10. Fluorescence: Terminally phospholinked nucleotides

NextSeq

1  75e2  150 bp

10e120 Gb

11. Electrical sensing

HiSeq (2500)

1  50e2  250 bp

10e1000 Gb

12. Inaccurate homopolymer detection

NovaSeq (5000/6000)

2  50e2  150 bp

2000e6000 Gb

13. Error rate: 0.01%

RS II

N50 20 Kb

0.5e1 Gb

Sequel

N50 20 Kb

5e10 Gb

MinIon

Up to 100 Kb

Up to 20 Gb

GridION

Variable

Up to 20 Gb

17. Error rate: 13%

PromethION

Variable

Up to 125 Gb

18. Error rate: 30%

Illumina

Pacific BioSciences

Oxford Nanopore

1,5,9,15

3,10,17

Note for the features 1. Sequencing by synthesis

9. Fluorescence: Reversible terminators

14. Error rate: 0.2% 15. Error rate: 0.2%e0.8%

3,11,18

16. Error rate: 1.8%

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TABLE 4.1 NGS and TGS technology basics.

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effective template generation for Illumina sequencing platforms. However, this is not an issue for other sequencing platforms. Read lengths vary by sequencing platform, ranging from 50 bp (short reads) to 100 Kb (long reads) (Table 4.1). Sequencing error rates range from 0.01% for the SOLiD method to 30% for Nanopore technologies (Table 4.1). The 454 GS-related platforms are now rarely used (da Fonseca et al., 2016). Nevertheless, these high-throughput platforms have allowed broad research communities to develop various strategies for the investigation of transcriptomes, and thus, understand genetic information flows from genome to phenome for functional characterization.

4.2.2 RNA sequencing RNA sequencing (RNA-seq) simply profiles everything from 50 - to 30 -ends of all transcripts with the production of either strand-specific or nonstrand specific reads (Morin et al., 2008; Parkhomchuk et al., 2009). The RNA-seq approach was initially called “whole transcriptome shotgun sequencing,” and is based on randomly primed cDNA and massively parallel short-read sequencing on an Illumina Genome Analyzer I. Library preparation starts with the synthesis of full-length, single-stranded cDNAs using modified oligo(dT) and template-switching primers by reverse transcription, followed by endto-end PCR amplification. The amplified products are then fragmented, and the fragments with 100e300 bp are selected for end-repair, dA tailing, and ligation with the Illumina sequencing adaptors. The ligated fragments serve as templates for PCR amplification to produce the final library for sequencing. Alternatively, libraries can be constructed with fragmented RNA, rather than cDNA (Wang et al., 2009). Unfortunately, fragmentation at either step introduces biases: RNA fragmentation is biased against both 50 and 30 -ends, whereas cDNA fragmentation remarkably favors the 30 -ends. To develop strand-specific RNA-seq (ssRNA-seq), Parkhomchuk et al. (2009) synthesized second-strand cDNA with dUTP rather than dTTP. The double-stranded cDNA was then fragmented and ligated with a Y-shaped adaptor. The second-strand cDNA was eventually removed with uracil N-glycosylase, an extremely efficient enzyme that selectively destroys uracil. Consequently, only first-strand cDNA is sequenced, which generates ssRNA-seq reads. Without a doubt, the ssRNA-seq method is work-intensive and requires several steps to complete, which lowers its practical utility. In addition, RNA-seq often requires 5e20  more reads to fully sequence a transcriptome when compared to traditional 50 or 30 -end sequencing methods (Rallapalli et al., 2014). Nevertheless, RNA-seq remains the most popular method for transcriptome analysis.

4.2.3 50 -end sequencing Profiling the 50 -ends of transcripts is usually achieved by enrichment of the 50 G(guanosine)-cap (Ozsolak and Milos, 2011). Traditionally, bacterial alkaline phosphatase is used to remove RNAs without a 50 -G-cap, whereas tobacco acid pyrophosphatase is employed to add a phosphate group at the 50 -end of transcripts. In preparation for a deep CAGE (cap analysis of gene expression) library, for instance, the first-strand cDNA is synthesized using random primers, and cDNAs with cap sites are trapped (Valen et al., 2009). Like the SAGE (serial analysis of gene expression) method (Velculescu et al., 1995), a linker that contains a recognition site for MmeI is ligated to the 50 -end of cDNA and then primed for the synthesis of the second-strand cDNA. Next, double-stranded cDNA is cleaved with MmeI, generating a 20 bp/21 bp tag for each cDNA. A second linker is added to the 30 -end of the digested tag, and the final library is prepared by PCR amplification and purified for sequencing without cloning. NanoCAGE and CAGEscan libraries were first described by Plessy and coworkers (Plessy et al., 2010), who used reverse transcription in combination with template-switching primers to produce 25 bp tags after EcoP151 digestion. The library preparation process involves many steps so that not many laboratories can handle them easily. In addition, the assignment of short tags (20e25 bp) to genes or genome regions is also challenging (Jiang et al., 2013). To date, the CAGE-based 50 -end sequencing methods have been heavily used by the FANTOM (functional annotation of mammalian genomes) consortium to profile alternative promoters, enhancers, and cisregulatory elements mainly in human and mouse (Lizio et al., 2019).

4.2.4 30 -end sequencing Procedures for library preparation that capture the 30 -ends of transcripts use either restriction enzyme digestion or chemical random fragmentation to yield polyA-associated short products for enrichment. Reverse serial analysis of gene expression (rSAGE) (Richards et al., 2006) and polyA tags (PATs) (Wu et al., 2011) with restriction digestion are two examples of NGS methods that employ restriction endonucleases. Regrettably, libraries that are prepared with endonuclease digestion often have missing data either due to the lack of enzyme recognition sites in transcripts or because product lengths are not optimal and uniform (Jiang et al., 2015). Examples of methods that use random fragmentation in library preparation include PATs, 3 PC (30 Poly(A) site mapping using cDNA circulation), 30 READS (30 Region extraction and deep sequencing), 30 T-fill, EXPRSS (Expression profiling through random sheared cDNA tag sequencing), PAS-seq (PolyA site

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sequencing) and PolyA-seq (Polyadenylation sequencing) (Derti et al., 2012; Hoque et al., 2013; Ma et al., 2014; Mata, 2013; Pelechano et al., 2012; Rallapalli et al., 2014; Shepard et al., 2011; Wilkening et al., 2013; Yao and Shi, 2014). Unfortunately, random fragmentation methods may produce noisy data (Ma et al., 2014). In addition, nonpolyadenylated RNAs and RNAs with short polyA tails may account for 20%e50% of all RNA (Katinakis et al., 1980). However, the tools that are currently used by the genome research community rarely profile these species, which contributes to an enormous gap in our knowledge of the role that they may play in the health and disease of humans and animals.

4.2.5 Isoform sequencing The RNA sequencing, 50 -end sequencing, and 30 -end sequencing methods described above usually yield short-reads for transcriptome analysis. Unfortunately, the reconstruction of full-length transcription isoforms using short reads is always a challenging task (Jiang et al., 2015). As such, teams from both PacBio, USA, and Nanopore, UK, have developed the socalled isoform sequencing (Iso-seq) methods. These platforms use single RNA molecules to generate full-length transcripts (van Dijk et al., 2018). The Iso-seq methods possess several advantages, such as single-molecule sequencing without amplification or fragmentation, a single read for entire exon-intron structure, high coverage of all splice sites of the original transcripts, the discovery of novel splicing forms and detection of allele-specific isoforms (Sharon et al., 2013; Tilgner et al., 2014). Because of library construction complexity and run depth, a potential dilemma may exist. If the Iso-seq libraries are not normalized, the transcriptome coverage might be relatively low due to the low number of reads per SMRT (single-molecule real-time sequencing) cell or flow cell. On the other hand, if the Iso-seq libraries are normalized, the true expression level per transcript cannot be obtained so that consequently, there are no ways to identify differentially expressed (DE) genes and/or isoforms among or between samples in comparison.

4.2.6 Single-cell RNA sequencing During the last 10 years, single-cell transcriptome analysis has become one of the best methods to decipher cellular heterogeneity, cell fates and states, subpopulation structures and cell-cell communications (Kulkarni et al., 2019). As such, there are at least 20 single-cell RNA sequencing(scRNA-seq) methods currently available (Chen et al., 2019). These scRNA-seq methods are classified into three groups: (1) full-length transcript sequencing (such as scWT-seq, Quartz-Seq, SUPeR-seq, Smart-seq, Smart-seq2, and MATQ-seq), (2) 50 -end sequencing (including STRT-seq and STRT/C) and (3) 30 end sequencing (such as CEL-seq, CEL-seq2, MARS-seq, CytoSeq, Drop-seq, InDrop, Chromium, SPLiT-seq, sci-RNAseq, Seq-Well, DroNC-seq, SCRB-seq, and Quartz-Seq2), respectively. Among them, six techniques were recently compared for sensitivity, accuracy, precision, power, and efficiency (Ziegenhain et al., 2017), including CEL-seq2 (cell expression by linear amplification and sequencing 2), Drop-seq (nanoliter droplet sequencing), MARS-seq (massively parallel RNA single-cell sequencing), SCRB-seq (single-cell RNA barcoding and sequencing), Smart-seq (SMART template switch-based sequencing) and Smart-seq2. The authors (Ziegenhain et al., 2017) used one million reads per cell as a saturation cutoff and found that Smart-seq2 is the most sensitive method, detecting a median of 9138 genes per cell, while the methods with the lowest sensitivities were MARS-seq and Drop-seq, which detected a median of 4763 and 4811 genes per cell, respectively. On the other hand, these six scRNA-seq methods profiled 17,000e21,000 expressed genes if cells were pooled. There were dramatic differences in accuracy among these six methods when transcript quantification was used as an indicator. In particular, detection accuracy was poor for low-abundance genes. As for precision, data were noisier when amplification was performed with PCR compared to in vitro transcription. When a combination of dropout rates and amplification noise were assessed, the SCRB-seq method exhibited the greatest power. Finally, Drop-seq was the most cost-effective technique because many cells are profiled simultaneously. Despite improvements, the currently available scRNA-seq methods have low sensitivities, efficiencies, and reproducibility in comparison to conventional RNAseq methods, thus producing data with more bias, noise, and uncertainty (Kulkarni et al., 2019). Unfortunately, the application of scRNA-seq methods in studies of cellular heterogeneity in farm animals has dramatically lagged behind research in humans and other model organisms.

4.3 Genome-wide profiling of ATS and APA sites 4.3.1 WTSS-seq and WTTS-seq design In order to standardize transcriptome profiling procedures and help the research communities further advance functional genomics in the postgenome sequencing era, Dr. Jiang’s laboratory at Washington State University has successfully developed two methods: whole transcriptome start and termini site sequencing (WTSS-seq and WTTS-seq) to profile either

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ATS or APA sites (Brutman et al., 2019; Zhang et al., 2018a, 2018b; Zhou et al., 2016, 2019). The starting materials are total RNA for both methods. For the former method, the library construction begins with the depletion of rRNA from total RNA, followed by purification of rRNA-depleted RNA, which is then mixed with 5 and 30 adapters for first-strand cDNA synthesis with reverse transcriptase. After synthesis, cDNA is treated with RNases I and H, so only single-stranded cDNA is left in the reaction, followed by size selection. The final step is to amplify the double-stranded cDNA with PCR, carry out the product size selection, and submit the library for sequencing. For the latter method, the assay involves RNA fragmentation, polyA þ RNA enrichment, first-strand cDNA synthesis by reverse transcription, and second-strand cDNA synthesis by PCR (Zhou et al., 2016). As such, WTTS-seq possesses several unique features. First, the WTTS-seq method adds adapters to both 50 - and 30 -ends of enriched polyAþ fragments during reverse transcription. Second, both polyAþ RNA and polyAþ cDNA are enriched using oligo(dT)25 beads and RNases I and H, respectively. Third, polyA-anchored primers are used to avoid PCR amplification detours and guarantee direct sequencing to the 30 -ends of transcripts. Lastly, overamplification is minimized, which maximizes transcriptome coverage. Data show that more than 99.9% of reads are derived from polyA sites when libraries are generated with the WTTS-seq method (Zhou et al., 2016). Furthermore, WTTS-seq data are highly correlated with RNA-seq data (Spearman’s rank correlation coefficient r ¼ 0.912) and detect more expressed genes when reads per million  0.2 cut-off points are employed (Zhou et al., 2016). A new protocol is also proposed to profile APA sites simultaneously from three types of RNAs:nonpolyadenylated RNAs, RNAs with short polyA tails, and polyadenylated RNAs with long polyA tails (Fig. 4.1).

4.3.2 Characterization of ATS and APA sites Once WTSS-seq and WTTS-seq libraries are sequenced, the raw reads are processed for end trimming, quality control, genome mapping, ATS or APA clustering and characterization, assessment of differential expression and pathway enrichment and others, for example (Zhou et al., 2019). Usually, a library that produces 5 e 10 million raw reads should be sufficient for processing and analysis. Read trimming occurs to these derived from WTTS-seq libraries because their 50 ends possess several Ts, which are complementary to the polyA tails. Read quality control can be carried out using the FASTX Toolkit version 0.0.13.1 (http://hannonlab.cshl.edu/fastx_toolkit/). If the Ion Torrent platform is used in sequencing, the TMAP version 3.4.1 (https://github.com/iontorrent/TMAP) is an appropriate tool for reading mapping against the reference genome. The ATS or APA sites are usually clustered using a 24-bp window. Based on their genome coordinates, ATS, or APA sites are then assigned to the intragenic and intergenic regions using the Cuff compare (v2.2.1) program (Trapnell et al., 2012). Intragenic ATS and APA sites can be located in exonic regions, extended exonic regions, intronic regions, and antisense strands. The ATS sites may also be located in 50 UTR (untranslated region) or further upstream region of a gene. Accordingly, APA sites are often derived from the distal sites (30 UTR) or the extended distal sites (downstream region of 30 UTR). Gene biotypes can be explored in the data analysis too, such as protein-coding genes, long noncoding genes, micro RNAs, pseudogenes, and small RNAs, which can be downloaded from NCBI databases for the species of interest. Last, the Metascape program (Tripathi et al., 2015) is a user-friendly tool for pathway enrichment. This program can take multiple lists of DEgenes to simultaneously pursue unique pathway analyses, and thus, understand gene networks under various conditions.

4.3.3 WTSS-seq and WTTS-seq: mutual validation As described above, WTSS-seq profiles the 50 -ends, while WTTS-seq collects information on the 30 -ends of transcripts. To validate that both methods yield similar results from the same samples, we examined RNA derived from six growing male and six growing female frogs (Zhou et al., 2019). At that time, we hypothesized that each method would produce extremely similar pathways that show differences between sexes. Based on the datasets, we first identified DE ATS and DE APA sites between males and females. These sites were then classified into two categories: upregulated in either males or females. As such, four lists of DE genes corresponding to two sets of ATS and two sets of APA sites were established for pathway enrichment. Among 100 top summary pathways (Fig. 4.2), Metascape analysis revealed that WTSS-seq and WTTS-seq methods yield the same sets of 51 and 37 “summary” pathways upregulated in males and females, respectively. In addition, 12 “summary” pathways were shared by both sexes. Certainly, there were minor differences between WTSS-seq and WTTS-seq methods. For example, DE APA sites produced five additional “summary” pathways than DE ATS sites upregulated in females, while DE ATS sites revealed two additional enriched “summary” pathways compared to DE APA sites upregulated in males. In addition, the WTTS-seq method did not identify the pathway called the mitochondrion organization in comparison to WTSS-seq in females (Fig. 4.2). However, the WTTS-seq identified one more pathway in males, chromatin organization, than WTSS-seq. Furthermore, gene sets involved in each pathway were not completely the same between WTSS-seq and WTTS-seq.

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AAAAAAAAAA...An Long polyA+ RNA AAAAA Short polyA+ RNA

Total RNA

PolyA- RNA rRNA rRNA depletion AAAAAAAAAA...An Long polyA+ RNA Short polyA+ RNA AAAAA PolyA- RNA A tailing AAAAAAAAAA...AnAAAAAAAAAA...An AAAAAAAAAAAAAAA...An AAAAAAAAAA...An

RNA without rRNA

Chemically Polyadenylated RNA

Fragmentation Fragmented RNA

PolyA enriched 3’ ends

5’ adaptorGrGrGr

AAAAAAAAAA...AnAAAAAAAAAA...An AAAAAAAAAAAAAAA...An AAAAAAAAAA...An PolyA+ RNA enrichment with oligo(dT25) beads AAAAAAAAAA...AnAAAAAAAAAA...An AAAAAAAAAAAAAAA...An AAAAAAAAAA...An Reverse transcription with switching and oligo(dT) adaptors AAAAAAAAAA...AnAAAAAAAAAA...An NVTTTTTTTTTT3’ adaptor

First cDNA Synthesis

AAAAAAAAAAAAAAA...An

5’ adaptorGrGrGr

NVTTTTTTTTTT3’ adaptor AAAAAAAAAA...An

5’ adaptorGrGrGr

NVTTTTTTTTTT3’ adaptor RNases I and H treatment to remove all RNA molecules First cDNA only

NVTTTTTTTTTT3’ adaptor PCR

Second cDNA synthesis

AAAAAAAAAA

5’ adaptorGrGrGr

NVTTTTTTTTTT3’ adaptor Size selection

cDNA

ATTS-seq library

FIGURE 4.1 Flowchart of a novel WTTS-seq assay. The starting materials are nonpolyadenylated, short- and long-polyadenylated RNAs plus rRNA. This novel assay involves rRNA depletion, in vitro polyadenylation, fragmentation, polyA þ RNA enrichment, first-strand cDNA synthesis by reverse transcription, and second-strand cDNA synthesis by PCR.

4.3.4 Advantages of WTTS-seq over RNA-seq Let us use WTTS-seq as an example to show its advantages over RNA-seq (Zhou et al., 2019). First, WTTS-seq, rather than RNA-seq, can directly measure the diversity and dynamics of alternative polyadenylation patterns specific to biological events. Second, WTTS-seq accurately detects an abundance of short transcripts, whereas biases against both 50 and 30 -ends occur in the RNA-seq assay, making short transcripts difficult to detect (Wang et al., 2009). Third, when genes overlap, only WTTS-seq reads can be clearly assigned to corresponding genes because they are strand-specific. Fourth, APA sites that extend from exon to intron, located in intron and expressed from the antisense strand, can be simply detected by WTTS-seq. However, these sites often remain unannotated in RNA-seq data. Fifth, RNA-seq detects more DE genes than WTTS-seq. The same situation was also observed by Wilkening and colleagues (Wilkening et al., 2013) in the development of their “30 T-fill” method that profiles genome-wide polyadenylation events. Basically, RNA-seq “exaggerates” DE gene identification, because it widens the distribution of transcriptomes and thus magnifies the fold changes for more DE genes. Therefore, some DE genes detected by RNA-seq are probably false positives. Lastly, WTTS-seq is

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FIGURE 4.2 Mutual validation of WTSS-seq and WTTS-seq. Both methods identified almost identical differences between males and females. Pathway enrichment was performed using the Metascape program (Tripathi et al., 2015). FeATS and FeAPA: upregulated pathways in females over males; and MaATS and MaAPA: upregulated pathways in males over females based on differentially expressed ATS and APA sites.

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more cost-effective than RNA-seq. Generally speaking, the WTTS-seq protocol and procedures are very similar to those used in the preparation of a library for RNA-seq, so library construction expenses should be similar. However, sequencing costs are significantly different. Based on our data analysis, we estimate that 5 to 10 million reads per WTTS-seq library should be sufficient for transcriptome analysis. In contrast, RNA-seq requires at least 30 million (75 bp) reads per library (Wang et al., 2011). Therefore, the cost of sequencing a library prepared by our WTTS-seq method is at least 67% cheaper than RNA-seq.

4.4 Genome to phenome via alternative transcriptome 4.4.1 Alternative transcriptome: bridges between genome and phenome Phenotypes, particularly those economically important traits in agriculturally important domestic animals, are complex, dynamic, and elusive in nature. Although genome communities have widely anticipated that a completely sequenced genome can help unravel all genetic secrets about an organism’s phenome, many challenges remain. This is why Rexroad and colleagues recently made a timely call for the Animal Genomics Research Community to focus on “genome to phenome: improving animal health, production and well-being” as a new USDA blueprint for Animal Genome Research 2018e2027 (Rexroad et al., 2019). This program aims at defining “the connection and causation between the genetic makeup of an animal (genome) and the totality of all phenotypes, or the observable physical or physiological traits or characteristics (phenome).” No doubt, this is one of the most challenging tasks in the postgenome sequencing era. An animal’s life, for example, begins with 1 cell (a fertilized egg) but ends with more than 250 types of cells supporting a dynamic phenome. During the developmental processes, in fact, the genome is pretty much fixed, but the phenome keeps advancing, evolving, and progressing with dynamics for diverse functions. The question is: how can a finite genome produce an infinite phenome during a lifespan? As the central dogma of molecular biology states “DNA makes RNA makes protein” (Crick, 1970), we believe that alternative transcripts are responsible for information flows from genome to phenome in response to internal, external and universal environments (Jiang, 2018).

4.4.2 Alternative transcriptome: interactions with gene biotypes Here, we use our studies on four species: cattle, chicken, rat, and X. tropicalis to demonstrate the usage differences in APA sites between coding and noncoding genes. The gene biotypes include four types of noncoding genes: lncRNA (long noncoding RNAs), miRNA (microRNAs), pseudogenes, and tRNA (transfer RNAs) and a type of coding gene: proteincoding genes. Gene biotypes significantly consistently affect the usage of APA sites among different species. For instance, tRNA and miRNA genes rarely execute alternative transcripts with only 1e1.23 and 1.15e1.57 APA sites per gene. The moderate use of alternative transcripts appears in pseudogenes and lncRNAs, which use 1.59e2.06 and 1.73e2.67 APA sites per gene, respectively. In contrast, protein-coding genes frequently execute alternative transcripts under different conditions, with an average of 3.44e5.81 APA sites per gene. As for the genomic locations, tRNA and miRNA genes do not use the intronic regions to express APA sites, but intronic APA sites account for 22.63%e46.37% and 22.36%e66.13% in lncRNAs and protein-coding genes, respectively. Usage of intronic APA sites in pseudogenes is dramatically different between mammals (3.83% in cattle - 5.33% in rat) and nonmammalian species (30.56% in chicken e42.72% in frog). The abundance and magnitude of APA site expression also vary considerably among gene biotypes, tissues/organs, developmental stages, and treatment conditions. Notably, average expressions of alternative transcripts in pseudogenes, miRNAs, or tRNAs, rather than the protein-coding genes, are often higher than any of the other five gene biotypes.

4.4.3 Alternative transcriptome: alteration under gene knockouts Protein kinase AMP-activated catalytic subunits alpha 1 (AMPKa1) and alpha 2 (AMPKa2) are two of the 50 adenosine monophosphate-activated protein kinase (AMPK) isoforms that serve as cellular sensors related to many biological events (Stapleton et al., 1996). Fiber size and muscle mass are significantly reduced (P < .05) in the soleus muscle from AMPKa1 KO (knockout) mice but are significantly increased in AMPKa2 KO mice (P < .05) in comparison to WT (wild-type) littermates (Fu et al., 2013). As such, we hypothesized that myogenesis in AMPKa1 and AMPKa2 KO mice is altered by the use of different types of APA sites, thus resulting in muscle phenotype changes (Zhang et al., 2018a). Total RNA was extracted from the gastrocnemius muscles and profiled using the WTTS-seq method. The AMPKa1 KO mice possessed more downregulated than upregulated DE-APA sites, while AMPKa2 KO mice had more upregulated than downregulated DE-APA sites. Interestingly, the downregulated DE-APA sites in the former model and the upregulated DE-APA sites in

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the latter model were rich in DE genes associated with muscle-related processes. These DE genes clearly explain why muscle area is reduced in AMPKa1 KO mice, while increased in AMPKa2 KO mice in comparison to WT mice. Most importantly, these data firmly suggest that alternative transcriptome profiling uncovers detailed information flows from genome to phenome. In brief, these results convince us that alternative transcripts serve as sensitive and powerful biomarkers that can be used to link genes to their functions via detailed biological processes.

4.4.4 Alternative transcriptome: responses to a high-fat diet Using our WTTS-seq method, we recently examined how binge feeding a high-fat diet (HFD) alters APA usage in the hypothalamus of male rats relative to control rodents fed a standard chow (Brutman et al., 2018). In this study, 763 of the 89,022 APA sites revealed in the species were determined as DE APA sites. Of these, 274 were downregulated, and 489 were upregulated (P  .01), respectively, in rats fed the HFD compared to rats fed the control chow. Based on DEgenes assigned to these DE-APA sites, we observed that the functional pathways were primarily related to neuron projection development and synapse organization. Phenotypically, HFD-exposed male rats showed characteristic hyperphagic feeding behavior by consuming significantly more calories than the controls in the early stage of the experiments, thus gaining an obese body weight relative to the controls in the later stage of the experiments. This implies that alternative transcriptome profiles can well explain information flows from genome to phenome induced by an obesogenic environment (Brutman et al., 2018).

4.4.5 Alternative transcriptome: the future of genome biology Understanding of the genome-to-phenome via alternative transcriptome processes will help us gain novel knowledge in many fields. For example, we will know (1) if each alternative transcript is a minimal functional unit in the genome, (2) if cellular environments trigger the use of different alternative transcripts or alternative transcripts drive cells to make phenotypic changes, and (3) whether or not alternative transcripts can sense instant changes related to DNA methylation, chromatin modeling, and genome reprogramming. The novel knowledge will certainly guide us to (1) develop specific lines/strains for maximization of desirable phenotypes, (2) establish crossbreeding programs for the gain of extraordinary hybrid vigor, (3) manage progeny tests for reduction of generation interval, (4) maintain long-term selection for consistent progress, and (5) discover novel biomarkers for diagnosis of diseases/pathogens to improve animal health, well-being, and production. This knowledge will help us to understand how animals use different sets of RNA alternatives to realize phenotypic adaption, justification, maintenance, memory, regulation, repair, resets, and transition. We anticipate that the flexibilities of alternative transcriptome usages will create novel phenotypes for future generations to use.

Acknowledgments This work was supported by the Eunice Kennedy Shriver National Institute of Child Health & Human Development of the National Institutes of Health under Award Number R21HD076845 and the National Institute of Food and Agriculture, United States Department of Agriculture under Award Numbers 2016-67015-24470 and 2020-67015-31733 to ZJ and by funds provided for medical and biological research by the State of Washington Initiative Measure No. 171 and the Washington State University Agricultural Experiment Station (Hatch funds 1014918) received from the National Institutes for Food and Agriculture, United States Department of Agriculture to ZJ.

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Chapter 5

RNA sequencing: a revolutionary tool for transcriptomics Minal Garg Department of Biochemistry, University of Lucknow, Lucknow, Uttar Pradesh, India

5.1 Introduction The much-celebrated central dogma published by Crick in 1958 defines the flow of genetic information contained in genes as DNA to RNA via transcription and finally into proteins via translation (Crick, 1958). Ever since their discovery, RNA molecules, collectively defined as transcriptome, are being recognized as key molecules in regulating biological activities within the cell. Chromatin and the transcriptional landscape are being examined to be precisely controlled by a range of overlapping sense and antisense, intronic and intergenic, and small- and large-sized (with interlaced exons) regulatory RNAs. Studies on these RNA molecules have greatly been expanded extensively over the past 2 decades. Many important functions are being assigned to them, which include regulation of epigenetic processes and subcellular organelles, chromosomal organization, integrity and remodeling, RNA turnover, protein-coding, and non-coding gene transcriptional control, translational regulation and genome defense in a cell and differentiation. Regulatory RNAs are identified to be greatly influenced by environmental signals. On account of the influence of environmental signals on regulatory RNAs, their characterization has important implications for understanding the geneeenvironment interactions. Transcriptome analysis, their identification, and quantification provides answers to many biological problems, including differences in the gene expressions in diseased/ healthy cells and during various developmental stages, pharmacogenomic responses, and understanding the evolution of gene regulation. This chapter outlines the power of sequencing RNA for the discovery and quantification of transcripts by a high throughput sequencing method called RNA-sequencing or RNA-seq. It provides the generic roadmap for the computational analysis of RNA sequences. The key analysis steps include preprocessing (experimental design, sequencing design, steps for quality control), core analysis (gene expression profiling, transcriptome profiling, and functional profiling), and advanced analysis (visualization and data integration).

5.2 Transcriptional landscape: regulatory RNAs Targeted deep sequencing may result in the precise identification of small and larger RNA molecules from different genomic locations in various cell types. The identification of different species of RNA molecules imparts a high degree of complexity to transcriptome. The mammalian transcriptional landscape is represented by the genes that express different classes of RNA molecules, including ribosomal RNAs, transfer RNAs, messenger RNAs, small nuclear RNAs, small nucleolar RNAs, long noncoding RNAs, microRNAs, PIWI-interacting RNAs, promoter-associated short RNAs, transcription initiation RNAs, and splice site RNAs (Fig. 5.1). Historically, RNA molecules were considered as simple, intermediate molecules between genes and proteins. Messenger RNA or mRNA is the most extensively studied RNA molecule. It acts as a transitory template and is directly translated into proteins via genetic code. Transfer RNA (tRNA) acts as an adaptor molecule, whereas ribosomal RNA (rRNA, highly expressed essentially in all the cells) provides a platform during protein synthesis. Genome-wide studies have shown the existence of regulatory RNAs outside the known boundaries of protein-coding genes that control the human ontogeny.

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FIGURE 5.1 Graphical representation of coding and non-coding RNAs on a mammalian transcriptional landscape.

New classes of common small RNAs (snRNAs) were later identified in the nucleus by biochemical fractionation. Many of these RNAs are identified as part of ribonucleoprotein (RNP) complexes. One of the classes of snRNAs is identified as spliceosomal RNAs that play a central role in RNA splicing. These snRNAs include U2, U4, U5, and U6 and are known to participate in RNAeRNA and RNAeprotein interactions. Recognition of the 5ʹ splice site and the branch point by U1 and U2 respectively, followed by the recruitment of U4, U5, and U6, displacement of U1 and interaction with U2 (through U6), as well as the 5ʹ and 3ʹsplice sites (through U5) are some of the key functions during the assembly and functions of spliceosomes. U11, U12, U4atac, and U6atac are identified as less abundant snRNAs, and along with U5 have a role in a variant “minor” spliceosome termed U12-type (Wang and Burge, 2008). Small nucleolar RNAs (snoRNAs) localized to the nucleolus are identified to chemically modify rRNAs, tRNAs, and snRNAs. Chemical modifications are essentially required for the maturation of tRNA and mRNA and during pre-mRNA splicing (requires modification of U2 snRNA), and thereby maintain normal ribosomal and cellular functions. The class of snoRNAs, box H/ACA subclass, are examined to guide pseudouridylation, whereas, box C/D subclass are known to guide the methylation of rRNAs, tRNAs and snRNAs (Henras et al., 2004; Meier, 2005). Disruption of the functions of snoRNAs leads to the loss of processing of 5.8S, 18S and 28S (or 25S in plants) rRNAs. Other sRNAs known as small Cajal body-specific RNAs (scaRNAs) are identified to be present in subnuclear structures called Cajal bodies that are known to process telomerase RNA. Another class of small RNAs, known as microRNAs (miRNAs) of 20e24 nucleotides in length, inhibit the translation and accelerate the degradation of target mRNAs by forming imperfect base pairing with the 3ʹUTRs of target mRNAs. These miRNAs can regulate a large number of target mRNAs, and reciprocally, many mRNAs contain target sites for a large number of miRNAs, and thereby control/regulate many physiological, developmental, and disease processes (Garg, 2015). Like miRNA, small interfering RNAs (siRNAs) are noncoding RNAs with similar in size of miRNAs. Both the classes of molecules posttranscriptionally regulate the gene expression by silencing the target mRNAs. Nevertheless, siRNAs are specific with one target mRNA, while miRNAs have multiple targets. Drosha, Dicer, and several Argonaute (AGO) proteins, the key genes and enzymes are involved in the biogenesis of dsRNA precursors. Cleavage of dsRNA precursors and its export from the nucleus to the cytoplasm is mediated by Drosha and Exportin-5, followed by its further processing by Dicer to small double-stranded RNA moieties. One strand of dsRNA (21e24 nucleotide) is loaded into the AGO proteins of the RNA-induced silencing complex (RISC). RISC is guided by small RNA strands to complementary RNA targets for its translational repression/destabilization (Zeng et al., 2003; Garg, 2015). Subclade of AGO proteins called PIWI (predominantly found in the nucleus) and PIWI-like proteins associate with another class of RNAs known as PIWI-interacting RNAs (piRNAs). piRNAs are small (26e30-nucleotide) RNAs and play an important regulatory role by epigenetically and post-transcriptionally silencing the transposons in germ cells (Girard et al., 2006). Transcription initiation RNAs (tiRNAs) and splice site RNAs (spliRNAs) are 17e18 nucleotides in

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length and are found in animals but not in plants. These classes of small RNAs, tiRNAs, and spliRNAs are associated with the initiation of transcription and splice sites, respectively. Their origin and functions are not completely defined, but they are suggested to play a role in modulating CCCTC-binding factor (CTCF) chromatin localization and nucleosome positioning. Other classes of less distinct RNAs include promoter upstream transcripts (PROMPTS), promoter-associated short RNAs (PASRs), and transcription start site-associated RNAs (TSSa-RNAs), which may play a role in RNA-directed transcriptional gene silencing (Kapranov et al., 2007; Han et al., 2007). Another noteworthy class of long non-coding RNAs (lncRNAs) are nonprotein-coding RNAs. They are nonpolyadenylated, around 200 nucleotides in length and are located intergenic (also known as large intergenic non-coding RNA [lincRNA]), intronic and antisense to protein-coding genes (Mercer et al., 2009). Loci that express lncRNAs exhibit indicative chromatin structure, promoter conservation, and regulation by conventional morphogens and transcription factors (Johnsson et al., 2014). Chromatin remodeling, regulation of epigenetic processes (X chromosome dosage compensation, and parental imprinting), transcriptional control, and posttranslational processing are the important functions assigned to them. Cell culture-based studies identify their functions in retinal and erythroid development, epidermal differentiation, breast development, regulation of apoptosis and metastases, and many other processes (Conesa et al., 2016). Northern blotting and quantitative PCR are low throughput methods and are restricted to quantify single transcript. Hybridization-based microarray methods are although high throughput and cheaper technologies for genome-wide quantification of gene expression but have a number of limitations, which include (i) limited accuracy toward the quantification of lowly expressed and very highly expressed genes, (ii) prior information of the sequences being interrogated, and (iii) appearance of cross-hybridization artifacts during the analysis of highly similar sequences (Shendure, 2008). On the other hand, sequence-based methods provide a direct determination of transcript sequence. Generation of expressed sequence tag (EST) libraries and dideoxy chain termination method of Sanger sequencing of complementary DNA (cDNA) is a low throughput method for the quantification of transcripts. Tag-based methods like serial analysis of gene expression (SAGE) and cap analysis gene expression (CAGE) quantify a number of tagged sequences and increase the accuracy; however, they fail to quantify the expression of splice isoforms. Next-generation sequencing (NGS) being high throughput sequencing of cDNA, provides a complete analysis of RNA and has revolutionized transcriptomics (Wang et al., 2009).

5.3 Transcriptome sequencing Transcriptome sequencing also called as RNA sequencing or RNA-Seq is a high throughput sequencing method, which allows (i) complete annotation of structures of transcripts (50 , 30 ends, as well as splice junctions), (ii) quantification of expressions of transcripts, (iii) measurement of extent of alternative splicing, and (iv) allele-specific expression (Battle et al., 2013; Lappalainen et al., 2013). Experiments should be carefully designed to achieve the objectives, which include the type of biological replicates, desired coverage across the transcriptome, and depth of sequencing. Typical RNA-Seq workflow consists of (a) sample preparation, which includes RNA isolation and its conversion to cDNA, (b) preparation of sequencing library, (c) sequencing it on NGS platform, and (d) bioinformatic data analysis (Fig. 5.2).

5.3.1 RNA isolation, reverse transcription and library preparation The total RNA sample isolated from biological replicates consists of rRNA, pre-mRNA, mRNA, and non-coding RNAs. The majority of the cellular pool consists of rRNA (95%), and its removal is important before library construction for successful transcriptome profiling; otherwise, it consumes the bulk of sequencing reads and limits the analysis of other less abundant RNA molecules. Enrichment of polyadenylated mRNA is by immobilizing it by allowing the binding of 30 poly-A tail of mRNA with the poly-T oligos (covalently bound to magnetic beads). Selective depletion of rRNA during isolation can be done by using commercially available kits, including RiboMinus (Life Technologies) or RiboZero (Epicentre). Following the denaturation and reannealing of double-stranded cDNA, preferential digestion of it by duplex-specific nucleases (digest the more abundant species, which re-anneal as a double-stranded molecule) selectively removes rRNA and other highly abundant mRNA transcripts like hemoglobin in the blood, insulin, immunoglobulins in mature B cells (Christodoulou et al., 2011). Nevertheless, different ribo-depletion protocols may result in variation in percent efficient removal of rRNA and differential coverage of small genes. Instead of selective depletion methods, recently developed methods for selective enrichment of regions of interest are also being adapted. Hybridization based

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FIGURE 5.2 RNA-sequencing workflow.

in-solution capture utilizes the use of biotinylated RNA transcribed from DNA template oligo libraries, which acts as baits and have complementary sequences to the regions of interest. RNA baits hybridize to molecules in the RNA-Seq library, and the bound complexes can be recovered using streptavidin-coated beads (Levin et al., 2009). For profiling small RNA molecules, including miRNA, siRNA, piRNA, key regulators of gene expression, a separate strategy is employed to isolate these molecules. Since these RNA molecules are lowly abundant, small in size, and lack polyadenylation, commercial kits are available, which involve isolation of small RNAs by size fractionation using gel electrophoresis. RNA bands of size 14e30 nucleotides are eluted from the gel and concentrated by ethanol precipitation. Silica spin columns can be selectively used to isolate small RNA molecules (Morin et al., 2010). For successful RNA-Seq experiments, isolated mammalian RNA should be of sufficient quality and quantity. Agilent Bioanalyzer can be used to determine the RNA quality by producing RNA Integrity Number (RIN) between 1 and 10. Highest quality samples with least degradation are designated with RIN of 10. Degraded RNA of poor quality has a RIN of less than six and affects the sequencing results via uneven gene coverage, 3e50 transcript bias and may lead to conclusions with errors. Sample integrity can be checked by gel electrophoresis, followed by analyzing the ratios of 28S to 18S ribosomal bands. Unfortunately, RNAs isolated from human autopsy samples or paraffin-embedded tissues are not of good quality (Thompson et al., 2007; Rudloff et al., 2010). Identification of cellular origins of disease, novel pathways, and previously unknown pathological sites are some of the advantages of sample heterogeneity. However, at the same time, the heterogeneous character of the biological source of RNA can seriously impact the estimations during transcriptome profiling. For isolating distinct cells, several methods can be employed, which include laser-capture microdissection and cell purification. Laser-capture microdissection is based on the separation of morphologically distinct cells under direct microscopic visualization (Emmert-Buck et al., 1996). Isolated RNA is of high quality, but the yield is poor, and therefore, requires amplification. Cell purification and enrichment protocols are based on the differential centrifugation and fluorescence-activated cell sorting (Cantor et al., 1975). Significant efforts have been put recently to develop single-cell RNA-Seq methods to uncover cell to cell variation in the expression of genes. Less than one pictogram of mRNA is present in 1 cell; however, most sequencing protocols require around four hundred nanograms to 1 microgram of input RNA. PCR amplification methods can compensate for the small quantity of RNA, but linear amplification of transcript is not possible. Besides, amplification biases based on the nucleic acid composition of different transcripts may alter its relative abundance in the sequencing library. Single cell-based methods are under development; nevertheless, the shift from microliter-scale reactions to nanoliter-scale reaction volumes with microfluidic devices may reduce biases observed during sample preparation (Shalek et al., 2013; Wu et al., 2014).

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High-quality RNA isolated from cells is then reverse transcribed into cDNA. Following reverse transcription, construction of sequence library involves amplification of randomly primed cDNA molecules or their fragmentation followed by ligation of sequencing adapters. The construction of standard libraries does not provide information about the transcription of the original strand (information about which strand is transcribed in original mRNA). Protocols for the conversion of RNA into cDNA should be such that libraries created are derived from each cDNA strand, which actually represents the parent mRNA strand and its complement. Sequencing followed by computational analysis of splice site orientation, open reading frame information (ORF) in protein-coding genes, and biases in coverage between 50 and 30 ends in eukaryotic genes may provide strand-specific information. For yielding strand-specific reads, strand information can be preserved by ligating adapters to single-stranded RNA in a predetermined direction or the first strand of cDNA (Lister et al., 2008). This strategy is not useful owing to coverage bias at 50 and 30 ends of cDNA molecules. Further to preserve strandedness and to distinguish second strand cDNA from the first strand during library construction, chemical labels such as deoxy-UTP (dUTP) can be incorporated during the synthesis of second-strand cDNA, which later can be removed by enzymatic digestion (Parkhomchuk et al., 2009). Preparation of poly-A libraries and the ribo-depletion libraries are the choice of quantifying coding RNA and noncoding RNA (including pre-mRNA, which has not undergone posttranscriptional modification), respectively. Library construction methods based on multiplexing enables the sequencing of multiple sequences together. The advantage of multiplexing is the generation of a large number of reads per sequencing run (e.g., generation of up to 750 million pairedend reads on a single lane of an Illumina HiSeq 2500), which is required for the analysis of complex samples. Introducing 6-bp indices, also called “barcodes,” to each RNA-Seq library of multiple samples helps to identify the sample from which the read originated. Adequate transcriptome coverage is possible for 2e20 samples (Birney et al., 2007; Blencowe et al., 2009). Thirty to 40 million reads can sufficiently detect transcripts and quantify gene expression of moderate to high abundance; however, up to 500 million reads are required for rare and lowly expressed transcripts. To control technical variability and differentiate it from biological variability during transcriptome profiling studies, External RNA Controls Consortium (ERCC) developed a set of RNA standards for microarray and RNA-Seq experiments. These universal RNA synthetic spike-in standards consist of 96 DNA plasmids with 273e2022 bp standard sequences inserted into a vector of approximately 2800 bp. RNA synthetic spike-in standards are added to sequencing libraries at different concentrations (Jiang et al., 2011; Zook et al., 2012) to assess coverage, sensitivity, and quantification.

5.3.2 Sequencing platforms Interpretation of sequencing data and downstream analysis are affected by the differences in the sequencing method and platforms. Next-generation sequencing (NGS) platforms, including the one developed by 454 Life Sciences (Roche) and Illumina (formerly Solexa sequencing), are currently available for high throughput sequencing of transcriptome/ DNA megabases (Metzker, 2010). These platforms form an attractive alternative for the analysis of transcription factor binding sites, the extent of genetic variation, DNA methylation patterns, quantitative and differential expression. These sequencing platforms use the parallel Sanger sequencing/ sequencing-by-synthesis method to simultaneously sequence tens of millions of sequence clusters (Bentley et al., 2008). Two important challenges during single-cell RNA-seq are (i) efficiency of cDNA synthesis may limit the detection, and (ii) amplification bias, which may reduce the quantitative accuracy. For controlling the amplification bias, unique molecular identifiers (UMIs) can be used as an internal validation control. These are random sequences that label individual molecules, provide an absolute scale of measurement and efficiently eliminate the amplification of noise problem. Owing to greater coverage and depth for the same fixed cost, Illumina has become the choice of a sequencing platform. An ensemble-based Illumina platform, which allows the sequencing of many identical copies of a DNA molecule via sequencing by synthesis approach for the identification of relative levels of RNA expression of genes (Bentley et al., 2008). DNA molecules are immobilized on the surface of a glass flow cell, and while using fluorescently labeled reversible terminator nucleotides, these molecules are clonally amplified. Small-sized miRNA molecules can be efficiently read without any misalignment or loss of read due to the observed sequencing error rate of less than 1% by Illumina. Low throughput Illumina MiSeq is a desktop sequencer. It offers simplified workflow, faster turnaround, generates around 30 million paired-end reads in 24 h, and therefore, good for transcriptome sequencing on a smaller scale. Of late, the Illumina HiSeq platform has set the revolution and dominates the sequencing industry. This platform has two flow cells, and each flow cell has got one to eight lanes, where each lane generates many millions of short reads. Every time during machine run, DNA samples in these lanes can be independently sequenced. It takes from one and a half days to 12 days to complete the sequencing depending upon the total read length of the library.

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PacBio, single-molecule-based platforms are based on single-molecule real-time (SMRT) sequencing. This method requires the use of fluorescently labeled nucleosides and a DNA polymerase for uninterrupted template-directed synthesis (Eid et al., 2009). It enables the detection of a distinctive pulse of fluorescence in a real-time manner as and when the base gets enzymatically incorporated into a growing DNA strand. Owing to the absence of the PCR amplification step in SMRT, this platform avoids amplification bias and provides improved uniform coverage across the whole transcriptome. It also facilitates the detection of novel transcript structures by providing long reads with average lengths of 4200 bp to 8500 bp (Sharon et al., 2013). Generation of longer sequencing reads lead to the accurate identification of transcripts with alternative splicing isoforms, and therefore, the PacBio platform is ideal for de novo transcriptome assembly. Nevertheless, a high error rate of 5% due to insertions and deletions (indels) may lead to loss of sequencing reads and misalignment due to imperfect matching of erroneous reads on comparison with the reference genome.

5.3.3 Analysis of the transcriptional landscape Continual evolution in the sequencing methods and protocols provides a high-resolution view of the transcriptional landscape. Gene expression profiling by RNA-seq facilitates the discovery of novel structures of genes, allele-specific expression, and alternatively spliced isoforms. At the same time, technical improvements in sequencing methods pose challenges toward bioinformatics/ computational analysis and interpretation of data in order to unravel the complexity of transcriptome (Fig. 5.3).

5.3.3.1 Raw reads Reads are produced following the sequencing of RNA samples in the lanes of flow cell on the NGS platform. FASTQformat files containing RNA-seq data/ reads are generated. FastQC tool can be used on raw reads obtained from the Illumina platform to check their quality. Raw reads are examined for GC content, duplication in reads in order to detect sequencing errors, presence of adapters, PCR artifacts, and contaminations. However, acceptable duplication, or GC

FIGURE 5.3 Roadmap for RNA-seq computational/ bioinformatic analysis.

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content levels are an experiment and organism-specific. Quality of reads generally decreases toward 30 end of reads, and hence, to improve mappability, FASTX-Toolkit and Trimmomatic are used to eliminate poor-quality bases, trim adaptor sequences, and discard low-quality reads (http://hannonlab.cshl.edu/fastx_toolkit/; Andrews, 2014; Bolger et al., 2014).

5.3.3.2 Read alignment Aligners/ read mappers are used to align the reads against the whole genome. Depending upon the choice of read mapper, 70%e90% of RNA-seq reads can be mapped to the human genome. The percentage of mapped reads is an indicator of overall sequencing accuracy. The number of reads produced per lane depends upon the concentration/ number of cDNA molecules. Summation of the total number of reads mapped to exons within each gene is a measure of the overall expression of each gene. Since many of the RNA-seq reads map across the splice junctions of the genome and hence, it is more difficult to map the reads compared to the mapping of DNA-seq reads. Owing to the inability to handle spliced transcripts, conventional read mapping algorithms, including BWA and Bowtie, are not recommended for mapping transcriptome data (Langmead et al., 2010; Li and Durbin, 2009). Instead of these conventional aligners, splice-aware aligners are used to map the RNA-seq reads. More commonly used RNA-seq aligner tools include STAR, TopHat, MapSlice, GSNAP, and RUM. These tools although are different in terms of memory utilization, speed, and performance but can efficiently recognize the differences between a read with short insertion and a read aligning across an exoneintron boundary (Trapnell et al., 2009; Wu and Nacu, 2010; Wang et al., 2010; Grant et al., 2011; Dobin et al., 2013). Tolerances can be set to allow a minimum level of mismatches in each alignment. While mapping transcriptome, reads may fall onto exons that are shared by different transcript isoforms of the same gene, and hence, a more significant number of multimapping reads are expected. Total mapping percentage is lowered down if the reads coming from unannotated transcripts are lost. Accumulation of reads at the 30 end of transcripts in poly(A)-selected samples is indicative of the poor quality of RNA quality in the starting material. Picard, RSeQC, and Qualimap are the tools to check the quality during mapping. GENCODE’s RNA-seq Genome Annotation Assessment Project3 (RGASP3) has systematically evaluated the performance of RNA-seq aligners. Alignment tools are different from each other on the basis of mismatch and gap placement, basewise accuracy, exon junction discovery, and alignment yield (Engstrom et al., 2013).

5.3.3.3 Transcript assembly Mapping of the reads to the reference transcriptome facilitates the quantitative analysis and prevents the discovery of unannotated and new transcripts. Mapped reads can be assembled into transcripts by aligning the reads to the reference genome. If transcriptome annotation is comprehensive as in the case of humans, reads can be directly mapped to a Fasta-format file containing all the transcript sequences correspond to all the genes of interests. Unspliced mappers like Bowtie can be used for faster mapping of the transcriptome but it does not allow the de novo discovery of the transcript. Transcript assembly can be de novo reconstructed by assembling the contiguous transcript sequences with the use of a reference genome or annotations. SOAPdenovo-Trans, Oases, Trans-ABySS or Trinity packages can be used for the de novo construction of transcriptome (Grabherr et al., 2011; Schulz et al., 2012; Hass et al., 2013; Xie et al., 2014). The standard method for transcript assembly does not exist; nevertheless, it is challenging to reconstruct the transcripts from short-read data. Short reads rarely span across many splice junctions, and hence, it is difficult to construct a full-length transcript using short reads. The number of computational methods/ algorithms are being evaluated by RGASP3 for transcriptome reconstruction. Quality of transcript assembly is majorly affected by (a) the nature of transcriptome, which is defined by the degree of polymorphisms, alternative splicing, gene complexity, and dynamic range of expression, (b) technological challenges, which include errors during sequencing, and (c) computational/ bioinformatic analysis, which include gene annotation and inference of isoforms (Steijger et al., 2013; Midha et al., 2019). The number of accepted mismatches, strandedness of the RNA-seq library, the length of sequenced fragments, and the length and type of reads (short/long) are some of the important parameters that need to be considered during mapping. Reads may span splice junctions, and therefore, it gets difficult to identify splice junctions correctly. In the presence of notable differences with the reference, sequencing errors, non-canonical junctions, and fusion transcripts, it becomes more challenging to determine the splice junctions. On account of these issues, use of a gapped or spliced mapper is recommended. One of the most popular spliced mapper used for RNA-seq analysis is TopHat. This tool utilizes a two-step method, which includes the mapping of unspliced reads to locate the exons, followed by splitting of unmapped reads and its alignment independently to identify exon junctions. Other mappers like GSNAP can identify single nucleotide polymorphisms/ indels; MapSplice and PALMapper identify non-canonical splice junctions; GEM is

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used for ultra-fast mapping; and STAR is recommended for mapping long reads (Trapnell et al., 2009; Jean et al., 2010; Wang et al., 2010; Wu and Nacu, 2010; Marco-Sola et al., 2012; Kim et al., 2013). Read assignment uncertainty arises when reads are mapped to multiple transcripts that represent multiple isoforms of a gene. In order to eliminate this problem, only those reads are considered, which map uniquely; however, this strategy is not good for the genes that lack unique exons. Cufflinks and MISO can construct a transcript based on the reads map to a particular isoform (Conesa et al., 2016). Counting the number of reads that map to full-length transcripts using computational tools like Flux Capacitor, Cufflinks and MISO allows quantification of gene expression levels (Trapnell et al., 2010; Katz et al., 2010; Griebel et al., 2012). An expression can be quantified using HTSeq by counting the number of reads that map to an exon. Variations due to sequence composition bias, library fragment size, and read length may lead to inaccurate quantification of gene expression. Reads per kilobase of transcripts per million mapped reads (RPKM) metric is used to normalize read counts within-sample by taking care of read length and library-size effects. Another within-sample normalized transcript expression metric known as fragments per kilobase of transcript per million mapped reads (FPKM) metric takes care of variances during paired-end reads (Mortazavi et al., 2008; Trapnell et al., 2010).

5.3.4 Differential gene expression analysis Gene expression experiments are normally done with an aim to quantify the transcripts for the differential gene expression analysis among different samples/ across different conditions. A number of statistical methods are developed to specifically analyze RNA-seq data. Discrete read counts across the replicates do not fit normal distribution but follow a Poisson distribution. However, it is difficult to define biological variability based on Poisson distribution, and this leads to high false positivity due to the underestimation of sampling error (Marioni et al., 2008). Gamma-Poisson distribution also known as negative binomial distribution accounts for additional variance or overdispersion (beyond the variance expected from random sampling) is able to best fit the read counts (RNA-seq data) across the biological replicates. As far as the sampling variance of small reads is considered, discrete distribution is not required for differential expression analysis (Anders and Huber, 2010). Cuffdiff, part of the Tuxedo suite of tools (Bowtie, Tophat, and Cufflinks), is popularly used to analyze RNA-seq data for differential expression analysis. In addition to this tool, other tools like DEGseq (based on the Poisson distribution), DESeq2 (uses negative binomial as reference distribution and provides its own normalization approach), edgeR (performs integrated normalization and differential expression analysis after the entry of raw reads and the introduction of possible bias sources to the model), Bayesian approaches include negative binomial models (EBSeq and BaySeq) are used. These approaches describe the differences among experimental groups and assign significance to differentially expressed transcripts. Data transformation methods take care of sampling variance of small read counts, create discrete gene expression distributions, and analyze data by regular linear models. NOISeq or SAMseq are nonparametric approaches that estimate null distribution for inferential analysis from the actual data alone. Careful observations of biological interpretations are important. It is also necessary to understand the model parameters and their limitations to derive meaningful and error-free biological conclusions. If sample availability is not the issue, then considering the tremendous drop in the price of RNA sequencing, a minimum of the three replicates per sample should be analyzed to leverage the reproducibility among the replicates. Biological replicates over technical replicates are preferred. No single method is competent enough for making analysis for all the datasets. The type of statistical method or even the version of the software package should be carefully selected because the choice of methods remarkably affects the outcome of the analysis (Conesa et al., 2016).

5.3.5 Alternatively-spliced transcript analysis Specific algorithms are used on RNA-seq data to detect changes in the expression of transcript isoforms originating from the same gene within the sample replicate. In one of the approaches, the detection of isoform expression is integrated with the identification of differential expression and the changes in the proportion of isoforms within the total expression of a gene are monitored. CuffDiff2 detects isoform expression and compares the differences among the expressed transcript isoforms, whereas BASIS directly detects differential expression of transcript isoforms (Zheng and Chen, 2009; Trapnell et al., 2012). Flow difference metric (FDM) analyses isoform by using aligned cumulative transcript graphs from mapped exon and junction reads while Jensen-Shannon divergence determines the difference (Singh et al., 2011). The statistical method that uses the rSeqDiff algorithm detects simultaneously the differential expression of isoform, as well as gene without splicing change. These methods limit the detection of isoforms with the short read RNA-seq data (Shi and Jiang, 2013).

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DEXseq and DSGSeq are used to identify alternatively spliced transcripts by comparing significant differences in the distributions of reads on exons and junctions of genes (Anders et al., 2012; Wang et al., 2013). Differential splicing can be detected with the help of DiffSplice, which identifies alternative splicing modules (ASMs) by using alignment graphs. These exon based approaches are more conveniently used for the identification of individual alternative splicing events if instead of the whole isoform, only specific exons that translate into functional/ regulatory domains are focused (Hu et al., 2013). The efficient display of multiple layers of information of transcriptome requires comprehensive tools with desirable features. A number of tools are used to visualize RNA-seq data, which includes ReadXplorer (at the level of reads), read pileup (at the level of processed coverage), and genome browsers like UCSC browser, Integrative Genomics Viewer (IGV), Genome Maps, or Savant are used for visualizing normalized and unnormalized (total count) data. RNAseq Viewer, although slower than IGV, displays reads on exons, transcripts, and junctions. Sashimi plots are used to display junction reads, and therefore, used to visualize differentially spliced exons. Sashimi, structure, and hive plots are used for visualizing splicing quantitative trait loci (sQTL). Visual comparison of signals on multiple samples can be done with the help of heatmaps.

5.3.6 Allele specific expression The ability of RNA sequencing to detect single nucleotide difference is exploited to sequence transcript reads across the heterozygous sites (maternal and paternal alleles). Differences in the transcript read sequences from the maternal, and paternal alleles give rise to allele-specific expression (ASE). Genetic variability, including single nucleotide polymorphism in the cis-regulatory/ upstream region of the gene and epigenetic changes, including methylation, histone modification, and genomic imprinting, are the potential mechanisms of allele specific variation in expressions. Thirty percent of the loci within an individual can be affected and incorporate allele-specific differences in the expression (Ge et al., 2009). ASE contributes to variable expressivity and its functional implications are variable disease penetrance and severity among individuals (Deng et al., 2014). For detecting ASE, reads covering the allele at heterozygous sites are retrieved, and either binomial test or Fisher’s exact test is applied. However, technical challenges encountered in ASE detection include sampling variance, alternatively spliced alleles, insertions, and deletions, overdispersion at extreme read depths, genotyping error, and read-mapping bias. Statistical tests like a beta-binomial distribution at individual loci take care of overdispersion (although it requires replicates) while the hierarchical Bayesian model approach makes global and site-specific inferences by analyzing reads across loci, as well as across replicates. Enhanced reference genome covering all SNP positions/ alternative alleles at polymorphic loci is constructed to take care of read-mapping bias and further improves the ASE detection (Satya et al., 2012).

5.3.7 Fused gene analysis Transcriptome mapping loses linearity when the chromosomes undergo rearrangements. It gives rise to fused genes/ novel isoforms. Its detection is a challenge because analysis needs much larger space. Reads cannot be uniquely mapped to the location of origin in the reference genome, and therefore, despite the use of advanced tools, it produces artifacts. Artifacts arise from the misalignment of read sequences due to homology, polymorphism, and or sequencing errors, and hence, its analysis requires the use of heuristic filters for postprocessing. The use of filters helps in the production of high-quality fusion candidates. Reads/ counts corresponding to the pairs of homologous genes, highly expressed genes (unlikely to be involved in gene fusions), and highly polymorphic genes are recommended to be carefully excluded/ filtered during analysis so that the events of interest are not lost. In the absence of control/ reference data sets, artifacts are identified by their appearance in a large number of unrelated datasets, after excluding their possibility of being representative of true recurrent gene fusions. Owing to the greater biological impact of chimeric sequences/ gene fusions (e.g. immunoglobulin [IG] rearrangements in tumor samples infiltrated by immune cells) over other forms of genetic variation, it is very important to successfully analyze them. Indicators of strong prediction strength are longer sequence length/longer reads and splicing. Longer reads/ sequence lengths increase alignment specificity, and thus, allow strong fusion-sequence predictions. Fusion isoforms normally follow splicing patterns of wild-type genes. Fusion boundaries and exon boundaries coincide with the splice sites, and hence, analysis of splicing sites increases prediction strength for the detection of fusion genes (McPherson et al., 2011).

5.3.8 Small RNA analysis RNA sequencing has been increasingly considered as a very popular technique that unravels many important aspects related to the biological roles of small RNAs (sRNAs). sRNAs include siRNAs, miRNAs, pi-RNAs, and other small

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regulatory RNAs, which are around 18e34 nucleotides in length. Contrary to regular RNA-seq libraries, sRNA-seq libraries are rarely deeply sequenced (2e10 million reads) due to a lack of complexity. After removing ligated adaptor sequences, read length distribution is computed. miRTools 2.0 use reads of 18bp to 30bp for prediction and profiling of sRNA. Burrows-Wheeler Aligner (BWA), STAR, Bowtie2 bioinformatic tools are used to align and map the sRNA reads to the transcriptome or reference genome. PatMaN and MicroRazerS aligners with preset parameter value ranges are specially designed to map short RNA sequences. Mapping can be performed with or without mismatches. Tools like miR analyzer and miRDeep are used for the analysis of miRNA sequences. miRNA sequencing reads are mapped to special databases like miRBase and mirWalk. miRBase and mirWalk are the repositories that contain known miRNA loci across many species and offer target prediction according to various algorithms. miR analyzer and miRDeep tools detect known miRNAs, which are annotated on miRBase. miR analyzer is able to predict novel miRNAs by using a machine-learning approach based on the random forest method. Novel miRNAs by miRDeep tool can be predicted from the RNA seq reads across the secondary structure of precursor miRNAs. Mapping is based on the properties of miRNA biogenesis for scoring the compatibility of the position and frequency of sequenced RNA. These tools are not only commonly employed for target prediction but also for quantifying expression and differential expression analysis (Hackenberg et al., 2011; An et al., 2013). A recent publication by Monga and Kumar provides a comprehensive review of applications of next-generation sequencing for computational identification of miRNAs, and their variants are omiRs, differential expression, miR-SNPs, and functional annotation (Monga and Kumar, 2019). Significant contamination of sRNA reads with degraded mRNA should be verified and ruled out. This can be done by checking the miRNA library for any unexpected read coverage over the body of highly expressed genes.

5.3.9 Expression quantitative trait loci analysis Biological information stored in the form of genotypic data can be integrated with the expression data obtained from RNA sequencing studies. This integration leads to the identification of genetic loci that exhibit variable gene expression. Such loci are known as expression quantitative trait loci (eQTLs). Variations in the expressions at loci contribute to the phenotypic variability of traits and differences in disease susceptibility across individuals. RNA sequencing studies made it possible to significantly examine a higher number of eQTLs compared to microarray studies. These eQTLs can influence the expression of genes in an allele-specific manner. They are located near transcriptional start sites and thus influence the expression of genes in cis (directly) or in trans (by affecting the expression of upstream factors that regulate the gene). The identification of eQTLs offers an understanding on the gene regulatory mechanisms. For improving the detection of regulatory variants, information on allele specific expression is integrated with eQTL analysis. Genotype data featuring millions of SNPs and expression data featuring tens of thousands of transcripts, obtained from high-throughput sequencing, require extensive computation to test the billions of transcript-SNP pairs for eQTL analysis. For facilitating data analysis, Matrix eQTL software is developed that utilizes either additive linear (least-squares model) or categorical (ANOVA model) to test the associations (Shabalin, 2012). These methods were used for studying RNA-seq data after normalization of total read counts. These models allow separate testing for each transcript-SNP pair to identify the association. Nonlinear models, including generalized linear, and mixed models, Bayesian regression, are also used to test associations. Bayesian methods are developed to test the effects of multiple single nucleotide polymorphisms on the expression of a single gene. Merlin, another model, detects eQTLs from related individuals using pedigree data (Abecasis et al., 2002; Servin and Stephens, 2007; Lee et al., 2008). Specific ribo-depleted RNA-Seq libraries are used to detect eQTLs that affect the expression of other RNA species, including various ncRNAs (long intergenic non-coding RNAs) (Kumar et al., 2013; Popadin et al., 2013). RNA-seq studies make it possible to profile alternately spliced isoforms of a gene and identify the variations in splicing. Variants that influence the quantitative expression of alternately spliced isoforms of a gene are known splicingequantitative trait loci (sQTLs) (Lalonde et al., 2011).

5.4 Future perspectives Genome-wide transcriptome profiling is routinely done in diverse cell types and cell states, including disease-specific cells, tissues, isolated cells, and cell-free biofluids (Marass et al., 2020). Recent developments in sequencing methods have made it possible to generate transcriptome data with a smaller amount of starting RNA as low as single cells. Protocols developed to generate and sequence longer reads that span multiple exoneexon junctions result in efficient quantification and detection of transcripts variants and alleles. The single-cell sequencing method with improved transcriptome coverage

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enables detailed analyses of gene regulatory mechanisms. Advancements in computational methods/ mapping tools are the desperate need of the hour to support novel applications and tackle the challenges for the accurate distinction between biological variations and technical variations. Progresses in RNA-seq methods facilitate the identification and validation of candidate biomarkers, mutations, SNPs, and thus allow early monitoring of disease, and provide greater insights into the logic behind the biological variations among different cell types and cell states.

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Chapter 6

Targeted genome editing: a new era in molecular biology Devlina Ghosh1, 2, *, Alok Kumar3 and Neeraj Sinha1 1

Centre of Biomedical Research, SGPGIMS-Campus, Lucknow, Uttar Pradesh, India; 2Amity Institute of Biotechnology, Amity University, Lucknow

Campus, Gomti Nagar Extension, Lucknow, Uttar Pradesh, India; 3Department of Molecular Medicine & Biotechnology, Sanjay Gandhi Postgraduate Institute of Medical Sciences, Lucknow, Uttar Pradesh, India *Corresponding author. e-mail address: [email protected]

6.1 Introduction Animal breeding and husbandry is a centuries-old tradition and dates back to the days of the beginning of human civilization. Since old times, farm animals are considered very valuable resources and may often act as models for physiological and pathological studies. Breeders have been altering the genomic characterization of animals by looking for the desired phenotypic traits and then selecting animals with the chosen features for further breeding for ages. Genomics is the branch of biological science that enables us to study the complete set of DNA in an organism (called the genome), it incorporates the usage of recombinant DNA, DNA sequencing methods, usage of bioinformatics in order to sequence, assemble, also analyze the structure and function of genomes. The study of animal genomics leads us toward better understanding the complexities of the genetic makeup and underlying mechanism to create better animals with preferred traits. There are several innovative techniques available nowadays to alter and improve the existing animals. Genes in our body carry the genetic information and act as templates to make structural proteins and all the necessary enzymes so that the living system can maintain the tissues and organs. There are four letters in our genetic code represented by A (Adenine), G (Guanine), T (Thymine), C (Cytosine). As we know, the genetic code is nearly universal, where Adenine always pairs with Thymine while Guanine pairs with Cytosine, and give rise to the double helix of DNA. Human beings have 23 pairs of chromosomes out of which there are 22 pairs of autosomes and one pair of sex chromosomes. Approximately 20,000 genes are organized in the chromosomes in a tightly packed manner inside the nucleus of each and every cell in the body. The expression of the gene in living cells are very tightly regulated so that all genes are not expressed at all times. Only the genes whose product is needed at a particular time is transcribed into mRNA, and in turn, translated into protein. This strict regulation at the gene level saves the resources of the cell so that there is no unnecessary wastage of energy and cell resources. Another interesting fact that is worth mentioning here is that, out of the total human genome, only 2% codes for proteins, while the rest 98% is noncoding sequences. The noncoding regions may contain noncoding RNAs, regulatory regions, repetitive DNA, etc. The repetitive DNA may be tandem repeats or interspersed repeats. Genomics is a relatively new stream of biology that basically focuses on studying the structure, function, evolution, and manipulation of the genome of an organism, as well as the interrelationship of different genes to the genome. It is the branch of science in which we study the whole genome (complete set of DNA with all genes) of organisms. Unlike in genetics, where we focus on a particular gene of interest, in genomics, the entire DNA content present in 1 cell of an organism is considered. Scientists, working in the field of genomics, endeavor to determine and study the complete set of DNA and do sequence mapping so as to understand the entire genetic composition of a living being. Such kind of study, eventually help us understand different diseases from the genetic perspective and to find out a probable cure by manipulating at the genetic level. It could have significant applications in clinical diagnostics and predictive testing. In simple words, genomics contains a massive amount of information about any particular organism in the form of DNA sequences. Every organism has a different genome size; for instance, the human genome comprises 3.2 billion bases of

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DNA while the mouse genome contains 2.5 billion bases. Human Genome Project (HGP) that was funded by National Institute of Health, USA, was an international endeavor to decode the human genome and determine the base pairs that make up the entire human genome; it was a decade long scientific effort that lasted from 1990 to 2003, spanning several prestigious universities and research organizations throughout the world. HGP, for the first time, enabled us to read and understand the complete genetic blueprint of the human genome. It was a very significant step in the field of genomics, so in the future, new ways can be developed to diagnose, treat, cure, or even prevent human diseases. Molecular biology, on the other hand, enables us to study biological molecules like DNA, RNA, protein, carbohydrates, etc. and their intricate interrelationships and tight regulation of their interrelationship. Bacteria and viruses have been the subject of earliest studies in molecular biology, due to their comparative ease of handling, simplicity in understanding the basic mechanisms. These earlier studies laid the foundation for unraveling the genetic code, as well as gene expression. However, studies on more complex organisms like Drosophila, zebrafish, plants etcetera were followed by the late 1960s and supported the underlying fundamental mechanisms. In bacteria, scientists unraveled the surprising and naturally existing mechanism of protection against viral invasion and led to the discovery of restriction endonucleases (popularly known as molecular scissors). That is how bacteria will cleave viral genetic material and thus stop viral propagation. These restriction endonucleases extracted from bacteria can cleave DNA precisely at particular recognition sequences (which are mostly palindromic, i.e., they are the same when read forward and backward). This discovery was immensely important and laid the stone for the development of gene-editing techniques. Animal genomics has led to the development of a variety of procedures to do genetic manipulation, study the genomic sequence and analyze the genomic information, which is contained in DNA that is the repository of complete genetic information and plays a pivotal role in the field of molecular biology. Some of the techniques involved are DNA cloning, DNA sequence study, macromolecular structure examination by Nuclear Magnetic Resonance, X-ray crystallography, amplification by a polymerase chain reaction, production of transgenic animals, etc. All these molecular biology techniques enable us to make desirable changes in the genetic material of any organism in order to get appropriate physiological changes. Genomics and molecular biology can go hand in hand and have emerged as a very powerful tool to study the gene functions and gene-specific manipulations to correct defective genes and also to introduce new functionality. Fyodor Urnov and his colleagues Edward Rebar, Philip Gregory, and Michael Holmes, while working at Sangamo Therapeutics, coined the term “gene editing” in 2005 (Urnov et al., 2005). Targeted genome-editing techniques help in doing site-specific, or rather, sequence-specific modifications in the DNA sequence. It encompasses several molecular biology techniques to manipulate genetic materials in order to create genetically modified organisms with desirable characteristics. In this chapter, we will discuss the techniques involved in targeted gene editing in detail to understand the evolution toward efficiency and precision. Targeted genome-editing techniques depend basically on special enzymes called endonucleases that can cleave DNA at specific sites, which can pave the way to edit the sequence by addition, deletion, recombination to bring desirable phenotypic changes. There are several genome-editing systems that are broadly used, for instance, homologous recombination, meganucleases, zinc-finger nucleases, transcription activator-like effector nucleases (TALENS), and Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR/Cas9) system. One of the earliest known ones is Homologous Recombination (HR), which is naturally occurring, almost universal phenomenon to produce new recombinations of DNA sequences during the process of meiosis, which gives rise to genetic variations in offspring and helps populations to adapt themselves evolutionarily in constantly changing environment. During this process, nucleotide sequences are exchanged between two identical or similar strands of DNA, thus promoting genetic modifications in the genetic makeup. Meganucleases are endodeoxyribonucleases as they possess endonuclease and deoxyribonuclease activity, and are characterized by a large recognition sequence, which is around 12e40 base pairs long. This feature imparts specificity to meganucleases as the probability of such a long sequence getting repeated in the genome is usually bleak. The high specificity of meganucleases enables them with a high degree of precision and comparatively lower cell toxicity than other naturally occurring restriction enzymes. Meganucleases are used to modify all genome types, whether bacterial, yeast, plant, or animal. ZFNs are comprised of zinc finger DNA-binding domain and an endonuclease, designed to bind and cleave DNA at specific positions. The domains can be modified to target specific desired DNA sequences, and thus, enables ZNFs to target unique sequences within a complex genome. ZFNs are site-specific endonucleases and artificially modified and contain two distinct protein domains; one is the DNA binding domain comprised of transcription factors and the zinc finger, the second one is the nuclease enzyme with a FokI restriction endonuclease. Together the two domains enable the ZFNs to bind at a defined site on the DNA sequence and cleave it. The double-stranded break in the DNA can be restored back by homologous recombination or nonhomologous joining.

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The mechanism of action of Transcription activator-like effector nucleases (TALENs) resembles that of ZFNs, as it involves artificial restriction enzyme created by fusing a TAL effector DNA binding domain to a DNA cleavage domain. The process starts with identifying the target sequence, and a corresponding TALEN sequence is engineered and inserted into the plasmid. The plasmid, containing TALENs with FokI endonucleases and TALE domains, is transfected in the target cell. After translation, functional TALEN comes into the picture, enters the nucleus, then binds and cleaves the target sequence. The discovery of the CRISPR/Cas9 system in recent years has offered an unprecedented way of modifying the genome of organisms in an efficient and cost-effective way. It is a part of the natural defense system in bacteria and archaea that have repetitive sequences in their genome called Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) to protect them against viral invasion. Researchers have been working on this system to use it for specific gene editing and have simplified it into a two-component system consisting of a short “guide” RNA that targets a specific sequence in the DNA and the cas9 enzyme, which has helicase and nuclease activity. The guide RNA can identify and hybridize a 20-bp protospacer in a particular genome. CRISPR-Cas9 can be easily adapted to target any genomic sequence by changing this 20-bp protospacer of the guide RNA. The guide RNA binds to the Cas9 enzyme, which then cleaves the target DNA. CRISPR-Cas9 system is a simple but very precise and strong technique to introduce alterations in specific portions of DNA to introduce genetic modifications and is considered the most reliable technique of gene editing so far. There are plentiful instances where gene-editing methods are used to understand better the gene function in genomes of human somatic cells, viruses, bacteria, animals, and plants. The implementation of gene editing tools has also been described in the field of plant biotechnology, animal breeding, and human health applications. In this chapter, we will discuss various targeted gene-editing techniques and their underlying mechanism in detail to understand the methods and how they are applied in research and to do gene manipulations so as to create desirable changes in the genomics, and in turn, in phenotypic features.

6.2 Homologous recombination Homologous Recombination (HR) is a naturally occurring, almost universal phenomenon to produce new recombinations of DNA sequences during the process of meiosis, which gives rise to genetic variations in offspring and helps populations to adapt themselves evolutionarily in constantly changing environment. As shown in Fig. 6.1, during this process, nucleotide sequences are exchanged between two identical or similar strands of DNA. One of the earliest methods of gene editing was HR, which is widely used by cells to precisely repair deleterious breaks that happen on both strands of DNA during interphase. Smithies et al., in 1985, first showed that specific modification of a human gene in vivo by HR between the endogenous locus and an artificially introduced piece of DNA is possible. Then on this process has been harnessed by scientists to design gene targeting constructs to undergo HR at a chosen locus to affect precise addition, deletion, or replacement of a particular DNA sequence. HR gives rise to edited alleles as recombination takes place between the host genome and double-stranded DNA donor molecules by stimulation introduction of a double-stranded break at the targeted sequence in the host genome. As sequences that are exchanged could be several kilobase-long, multiple genome modifications can be produced concurrently at a single locus. Method of homologous recombination, may be demarcated for creating: (i) alleles with simple sequence changes or in-frame additions, (ii) conditional alleles in which recombinogenicloxP sites flank exons and (iii) knockin/knockout alleles that express a reporter protein from an endogenous locus (Hoshijima et al., 2016). HR is one of the conventional gene targeting methods, which have been used by embryonic stem cells to develop more sophisticated methods that can enable allele-specific manipulation in zygotes (Gurumurthy and Lloyd, 2019). Several

Sister Chromatids

Crossing Over

Recombinants

FIGURE 6.1 Mechanism of homologous recombination: the two sister chromatids cross over and generate variation in resulting chromosomes.

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mouse models have been developed successfully using HR manipulation. The mouse has several biological features that make it the most commonly used animal model in biomedical research for understanding human disease mechanisms; these features include short life cycle, gestation period and lifespan, as well as its high fertility and propagation efficiency (Silver, 2001). This technology allows researchers to use the molecular tools to functionally understand mouse model and another genome with more reliability and specificity, as well as to make new mouse models using faster, simpler, and less costly techniques. HR also has a prominent role in double-stranded break repair. There are two key pathways worth mentioning, one is the homology-directed repair (HDR), and the other one is non-homologous end joining (NHEJ), involved in double-stranded break (DSB) repair. Single-stranded oligodeoxyribonucleotide (ssODN) facilitated homologous recombination repair is usually used for site-directed genome editing in animals, with important scientific and applied value. RNA-guided nucleases can create DNA double-stranded breaks (DSBs) to achieve genome editing through DSB repair. Out of the two main repair pathways to fix the break, homology-directed repair (HDR), is restricted to the S/G2 phases of the cell cycle and particularly less frequent. Accurate genome-editing applications depend on HDR, with the abundant c-NHEJ formed mutations presenting a barrier to achieving high rates of precise sequence variations (Danner et al., 2017).

6.2.1 Scientific/clinical significance During cell division, homologous chromosomes are vital in the processes of meiosis and mitosis. They permit the occurrence of recombination and random segregation of genome from parent cells into progeny. During the meiosis process of recombination produces new combinations of alleles in the offspring. Through different generations, a chromosome is not fixed, but rather it is very flexible, having several different combinations of alleles. As a result, nonfunctional/less functional alleles are cleared from a population. We can infer the significance of this process, as in its absence, one deleterious mutant allele would trigger an entire chromosome to be abolished from the population. However, recombination allows the mutant allele to be separated from the other genes on that chromosome. The defective allele can be removed from the population by negative selection. In addition to playing a key role in meiosis, recombination has two additional advantages for sexual species. It makes novel combinations of alleles along chromosomes, and it limits the consequences of mutations mainly to the region around a gene, not the entire chromosome. Under gene targeting techniques, homologous recombination can modify an endogenous gene and can be used to delete a gene, remove exons, add a gene, and modify individual base pairs (introduce point mutations). HR has been used on embryonic stem cells to knock out or knock in a specific gene and to generate transgenic mice. Although a classical kind, this method is very time consuming, labor-intensive, and much expensive to work out, and is feasible on small subsets like yeast and mouse (Hatada et al., 2000; Gaj et al., 2016; Smith et al., 2019).

6.3 Endonucleases/zinc-finger nucleases ZFNs are comprised of zinc-finger DNA-binding domain and an endonuclease, designed to bind and cleave DNA at specific positions. The domains can be modified to target specific desired DNA sequences, and thus, enables ZNFs to target unique sequences within a complex genome.The most common DNA binding motif present in eukaryotes and symbolizes the second most frequently encoded protein domain in the human genome is the Cys2eHis2 zinc-finger. A single zinc finger consists of nearly 30 amino acids in a conserved bba configuration (Beerli and Barbas, 2002). Each ZFN comprises of two functional domains; one is a DNA-binding domain made up of a chain of two-finger modules, each recognizing a unique hexamer (6 bp) sequence of DNA. Two-finger modules are joined together to give rise to a zinc-finger protein, each with a specificity of
24 base pairs. The second domain is a DNA-cleaving domain, including the nuclease domain of Fok Right ZFP Fokl Double Stranded DNA Fokl Left ZFP

DNA Cleavage Site.

FIGURE 6.2 ZFN composed of zinc fingers that recognize triplets and the FokI nuclease that acts as a dimer, cleaving in the region between two distinct target sites.

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I. As Fig. 6.2 shows, when the DNA-binding and DNA-cleaving domains are glued together, a highly specific pair of “genomic scissors” is generated. The underlying mechanism involves an array of zinc-finger binding domains that recognize the target DNA sequence and zinc-finger nucleases composed of Fok I endonuclease responsible for catalytic cleavage of DNA. A donor DNA template provided in trans can then be integrated at the site of strand break to give rise to transgenic DNA sequence. However, the absence of donor DNA can result in disruption of the open reading frame as insertions/deletions are introduced. In eukaryotes, zinc fingers are the most common DNA binding domain, usually comprised of w30 amino acid units that interact with nucleotide triplets. In recent years researchers have designed zinc fingers that recognize all of the 64 possible trinucleotide combinations, and by threading different zinc-finger moieties, custom ZFs can be created that specifically recognize any particular sequence of DNA triplets. Typically each ZF can recognize three to six nucleotide triplets. Also, the nuclease can only function as dimers. Therefore pairs of ZFs are needed to target any specific site so that one recognizes the upstream sequence and the other one for the downstream sequence to the target site that needs to be modified.

6.3.1 Scientific/clinical significance ZFNs have several advantages that make them a popular tool for gene modification. They can participate in the rapid disruption or fast integration into any genomic locus. Mutations that arise due to ZFNs are permanent in nature and heritable too. This tool works in different mammalian somatic cell types. Also, the gene edits can be introduced in a single transfection experiment. Using this method, knockout or knock-in cell lines may be created in a short time frame of 2 months. ZNFs may be used in optimizing cell lines by generating ones that produce higher yields of proteins or antibodies. Cell-based screening techniques are of special interest to scientists in various research methodologies and ZFNs can be useful in creating knock-in cell lines with promoters, fusion tags, or reporters integrated into endogenous genes. Fyodor Urnov and his team at Sangamo therapeutics were the first ones to apply gene-editing techniques, and the therapeutics advanced to a clinical trial in 2009 with a zinc-finger nuclease (ZFN)-based application on SB-728-T for HIV (Urnov et al., 2005). ZFNs have been used to create many genetic disease models, clinical trials of CD4þ human T cells with disruption of the CCR5 gene to serve as a potential treatment of HIV/AIDS. Despite being advantageous, due to lower specificity, ZNFs may lead to off-target binding and cleavage of DNA sequence, resulting in undesired genetic modification, which may be detrimental for the cell itself. Also developing target specific ZNFs are time-consuming and costlier to pursue (Carroll, 2011). Also, the use of programmable endonucleases has greatly increased the feasibility and ease of editing the mouse genome. Very recently, interesting findings came into the picture, which underlined the role of ZFNs in the therapeutic aspect, by employing AAV2/6 delivery of murine-specific ZFNs in vivo, stable levels of hFIX were detected in the blood of mice, which was injected with the albumin ZFNs, and hF9 transgene donor. It may be of crucial significance to determine if a single coadministration of ZFN and donor AAV vectors is sufficient to enable therapeutic and potentially lifelong production of the clotting factor for the treatment of Hemophilia B (Laoharawee et al., 2018). More recently, researchers have designed four-finger ZFNs that recognize an endogenous target site within the IL2Rg gene underlying the human X-linked disease, severe combined immune deficiency (SCID), and used them for ZFN-mediated gene targeting to attain very efficient and permanent alteration of the IL2Rg gene in human cells. This could be a breakthrough in the ZFN-based therapeutic approach in human diseases (Urnov et al., 2005). Despite its foray into the clinical aspect, it must be highlighted that ZFN-based approaches as a form of gene therapy are still at its infancy. There are numerous issues, like efficient gene delivery into the targeted cells and immune response to ZFNs, etc. that need to be focused on very thoroughly and carefully before ZFNs can be considered for human therapeutics. Gene-editing technology based on ZFNs has a number of drawbacks, which include the complexity and high expense of protein domains construction for each particular genome locus, and there are always chances of inaccurate cleavage of the target DNA due to single nucleotide substitutions or incorrect interaction between domains.

6.4 Transcription activator-like effector nucleases (TALENS) Research studies on plants, animals, and human genomes have opened up incredible opportunities for acquired data usage in biotechnology and medicine. However, just knowing the nucleotide sequences is not sufficient in order to understand the

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Right TALEN

Fokl

Double Stranded DNA

Fokl

Left TALEN FIGURE 6.3 Mechanism of TALENs.

functional implications of particular genomic elements and their function in phenotype determination and disease pathogenesis. Then there was Human Genome Project (1990e2003), which was a global scientific endeavor with the objective to decipher the precise sequence of nucleotide bases in the entire human genome. This opened up a rapidly evolving gateway allowing genomic DNA sequences manipulation, visualization, and regulation of gene expression. In 2011, Nature Methods designated the methods of detailed genome editing, comprising the Transcription activatorlike effector nucleases (TALEN) system (Baker, 2012). Bacteria belonging to the Xanthomonas genus, which basically are pathogens of crop plants, such as rice, tomato, pepper, etc. are involved in the history of this system’s development. These pathogenic bacteria cause substantial economic damage to agriculture, which was the motivating factor for their in-depth study. It was found that these bacteria secrete effector proteins (transcription activator-like effectors, TALEs) into the cytoplasm of plant cells, which disturbs the activities in plant cells, and in turn, increases its vulnerability to the pathogen. Further research on the action mechanism of the effector protein disclosed that they could bind to the host DNA and activates the expression of their target genes via imitating the eukaryotic transcription factors. As evident from Fig. 6.3, the mechanism of action of TALENs looks like that of ZFNs, as it encompasses artificial restriction enzyme created by fusing a TAL effector DNA-binding domain to a DNA-cleavage domain. Although unlike ZFNs, TALENS are more site-specific with less off-target effects as they can tackle one nucleotide at a time rather than three.The DNA-binding domain was demonstrated to consist of monomers, each of them binds one nucleotide in the target nucleotide sequence. If we go into the detailed mechanism, the process initiates with identifying the target sequence, and a corresponding TALEN sequence is plotted and inserted into the plasmid. Similar to ZFNs, TALE motifs are linked with FokI endonuclease that needs dimerization for the cleavage to occur. The plasmid, containing TALENs with FokI endonucleases and TALE domains, is transfected in the target cell. After translation, functional TALEN comes into the picture, enters the nucleus, then binds and cleaves the target sequence. TALEN construct may also be delivered in the cell as mRNA, and this prevents genomic integration of TALEN expressing proteins and improves the success of introgressive hybridization during gene modification. Theoretically, a double-stranded break (DSB) can be introduced in any region of the genome with known recognition sites of the DNA-binding domains by artificial TALEN nucleases. However, there has to be one T before the 50 - end of the target sequence, which is the only limitation to the selection of TALEN nuclease sites. However, site selection may be done in most cases by changing the spacer sequence length The crystal structure of TAL effector PthXo1 bound to its DNA target (Mak et al., 2012). Similar to ZFNs, the protein DNA association provides specificity to TALENs. And in the case of the latter, a single TALE motif recognizes one nucleotide and a collection of TALEs may associate with a longer sequence. It is important to mention that the activity of each TALE domain is limited to only one nucleotide and does not impact the binding of adjoining TALEs. Thus designing TALEs is relatively easier than ZFNs. TALENs are typically comprised of 18 repeats of 34 amino acids. These repeats have variability at 12 and 13 amino acids called the Repeat Variable Diresidue (RVD). The DNA-binding specificity of TALENs is conferred by a DNA-binding code mediated by the RVD. A TALEN pair needs to bind on opposite sides of the target site, separated by a “spacer” ranging from 14 to 20 nucleotides. As FokI requires dimerization for activity, this offset design is essential. However, such extremely long DNA-binding site (around 36 bp) is not found so frequently in genomes. Some degeneracy has been reported in RVD-DNA-binding site (Bogdanove and Voytas, 2011), so far there is very little evidence of mismatch tolerance or off-target activity. In a recent research study, an immune pluripotent stem (IPS) cell line was modified with an extremely active TALEN, and no mutagenic activity was noticed at other genome sites homologous to the target site (Park et al., 2014).

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Although the composition of TALEN involves reengineering a new protein for every target, TALEN design has been simplified by the accessibility of modules of repeat combinations that reduce the amount of cloning.

6.4.1 Scientific/clinical significance It is of immense advantage that Transcription activator-like effectors (TALEs) are significant genomic implements with customizable DNA binding motifs, which can do locus-specific alterations. In a zebrafish model, TALE Nucleases have been effectively used in order to do targeted mutations by repair of double-stranded breaks (DSBs), which could be done either via non-homologous end joining (NHEJ) or by homology-directed repair (HDR) and homology-independent repair in the company of a donor template. TALENs enable high binding specificity in comparison to other customizable nucleases and possess fewer sequence constraints in targeting the genome, with comparable mutagenic activity (Ma et al., 2016). Due to fewer off-target cleavages, and thus, lesser undesirable mutations, TALENS have been used in various clinical applications; for instance, allogeneic engineered T-cell therapy has been used successfully to treat two young children suffering from acute lymphoblastic leukemia. It has been used to generate knock out/knock-ins into the target sequence. TALENS have also been extensively used to modify plant genome, to enhance the yield, flavor, and other properties of economically important crops and also to produce biofuels. The DNA binding region of a TAL effector may be collectively joined with the cleavage domain of a meganuclease to generate a hybrid design combining the comfort of engineering and very specific DNA binding activity of a TAL effector with the low site frequency and specificity of a meganuclease. However, the process is very expensive and time-consuming (Zhao et al., 2016; Guha and Edgell, 2017). Methods discussed so far in this chapter for gene editing like ZFNs and transcription activator-like effector nucleases (TALENs), recognize their genomic DNA target through protein-DNA interactions, and therefore, to edit a new DNA site of interest, researchers had to engineer a new protein. The CRISPR-Cas9 system, an RNA-guided genome-editing tool, greatly facilitates genome editing because it can be reprogrammed quickly and inexpensively using readily available synthetic RNA molecules, and the technique is further discussed below.

6.5 CRISPR-Cas9 The foundation stone for genetics was laid by the work of Gregor Mendel during mid 19th century when he did detailed experimentation with plants, especially the pea plant, to see how the breeding variations bring about phenotypic changes in the progeny plants. Mendel’s scientific observations were published in 1866, which documented observations in around 10,000 plants spanned around 10 years to note targeted modifications in living cells that are brought in by breeding for desirable changes in phenotype. In current times, researchers in the biomedical field who are dedicatedly working on the gene therapy aspect, focus on therapeutic and medical applications directed toward finding a cure for particular disease conditions, especially inheritable diseases. Cancer has been one of the conditions that has attracted the attention of scientists worldwide, and gene editing has opened up a fascinating way of finding a therapy/cure for it. Paul Berg, in 1972, (Berg, 2008) pioneered the field of genetic engineering when, for the first time, he combined genes from two different organisms together and paved the way for the idea of recombinant DNA technology. It was a historic achievement when the E. coli genome was combined with genes of SV40 virus and bacteriophage. Since then, this science has achieved remarkable success, as the molecular basis of genetic mechanisms and phenomena can now be replicated in vitro. Research studies in the field of molecular genetics and biochemistry of bacteria, viruses, and other microorganisms have permitted the development of methods to manipulate DNA, create various vector systems and methods for their transport to the cell. Recombinant DNA Technology has proven to play a very significant role in the production of vaccines, several therapies like human insulin, interferon, and hormones etcetera. This technology is also used to generate clotting factors for the treatment of hemophilia and developing gene therapy. Since then gene-editing techniques have come a very long way, the most recent one that grabbed worldwide attention is CRISPR/Cas9, although considered a very young field and came into existence when scientists came through bacteria that have repetitive sequences in their genome called Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR), which has opened up tremendous opportunities and avenues of gene engineering and editing in the field of molecular biology and biotechnology. In this chapter, we will focus on different important aspects of CRISPR-Cas9 and will discuss its emerging role in genome editing and modifications.

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CRISPR-Cas9 system is a simple but very precise and strong technique to introduce alterations in specific portions of DNA to introduce genetic modifications and is considered the most reliable technique of site-specific gene editing so far.

6.5.1 Origin of CRISPR-Cas9 CRISPR-Cas9’s existence first came into the picture in as early as 1987, when an unusual repetitive DNA sequence, which was subsequently defined as a CRISPR, was discovered in the E. coli genome during an analysis of genes involved in phosphate metabolism (Ishino et al., 2018). Functional classification of CRISPR-Cas systems was an acute step toward recognition of a link between CRISPRs and the associated Cas proteins, which were initially thought to be involved in DNA repair in hyperthermophilic archaea, Francisco Mojica, a scientist at the University of Alicante in Spain, first discovered CRISPR in archaea (1990) and later in bacteria (2005) (Ishino et al., 2018). CRISPR-Cas9 naturally exists in a bacterial system as a defense system against infecting bacteriophages and plasmid transfer. The bacteria capture scraps of DNA from invading viruses and use them to create DNA segments known as CRISPR arrays. The CRISPR arrays provide the bacteria with genetic memory to detect the viruses (or closely related ones). If the outbreak of a virus occurs again, the bacteria yield RNA segments from the CRISPR arrays to target the viruses’ DNA. The bacteria then use Cas9 or a similar enzyme to cut the DNA apart, which incapacitates the virus. Streptococcus thermophilus bacteria were sequenced and studied by Bolotin, who then revealed an unusual CRISPR locus (Bolotin et al., 2005). Soon researchers began to fill in the gaps on how the CRISPR/Cas system inhibits the invading phage. John van der Oost and colleagues provided the first piece of critical information and showed that in E. coli, spacer sequences, which originates from phage, are transcribed into small RNAs, called CRISPR RNAs (crRNAs), that escort Cas proteins to the target DNA (Brouns et al., 2008). Sylvian Moineau and colleagues confirmed that CRISPR-Cas9 creates double-stranded breaks in target DNA at defined positions (Magadán et al., 2012). They also established that Cas9 is the only protein required for cleavage in the CRISPRCas9 system. This is a unique characteristic of Type II CRISPR systems, in which interference is facilitated by a single large protein (here Cas9) in combination with crRNAs. Jennifer Doudna and Emmanuelle Charpentier in 2007 filled the final piece to the puzzle in the decoding mechanism of natural CRISPR-Cas9-guided interference. They performed small RNA sequencing on Streptococcus pyogenes, which has a Cas9 containing the CRISPR-Cas system. They revealed that in addition to the crRNA, a second small RNA exists, which they called trans-activating CRISPR RNA (tracrRNA) (Deltcheva et al., 2011). They showed that tracrRNA forms a duplex with crRNA, and this duplex guides Cas9 to its targets. Zhang lab, in 2013, published the first method to engineer CRISPR-Cas9 to modify the genome in mouse and human cells (Hsu et al., 2014). They were first to successfully adapt CRISPR-Cas9 for genome editing in eukaryotic cells (Cong et al., 2013) Zhang and his team engineered two different Cas9 orthologs (from S. thermophilus and S. pyogenes) and demonstrated targeted genome cleavage in human and mouse cells. They also showed that the system could be programmed to target multiple genomic loci, and could also drive homology-directed repair. Researchers from George Church’s lab at Harvard University testified similar conclusions in the same issue of Science (Mali et al., 2013).

6.5.2 Underlying mechanism Many bacteria have a distinctive feature in their chromosome that includes repetitive sequences that flank unique sequences, along with these arrays, there were Cas genes typically located nearby that encode a protein and is a part of the adaptive immune system against invading viruses. Thus CRISPR is an array of short repeated sequences separated by

Matching Sequence

Cas 9 Guide RNA

Double Stranded DNA PAM Sequence FIGURE 6.4 CRISPR/Cas9 system, underlying mechanism.

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spacers with unique sequences. This naturally occurring phenomenon has since been used by researchers to do modifications and gene editing in controlled conditions. The underlying mechanism, as shown in Fig. 6.4, is as follows, a small piece of RNA with a short “guide” sequence that attaches to a specific target sequence of DNA in a genome is created. The guide RNA recognizes and hybridizes a 20-bp protospacer in the genome (Gupta and Musunuru, 2014). CRISPR-Cas9 can be easily adapted to target any genomic sequence by changing this 20-bp protospacer of the guide RNA. The guide RNA binds to the Cas9 enzyme. Like in bacteria, the altered RNA is used to identify the DNA sequence, and the Cas9 enzyme introduces incisions in the DNA at the targeted location. Once the DNA is changed, researchers use the cell’s self DNA repair machinery to modify (add or delete pieces of genetic material, or to make changes) the DNA by replacing an existing segment with a customized DNA sequence (Nuñez et al., 2015). As discussed before in this chapter, CRISPR/Cas systems give bacteria and archaea a unique adaptive immunity against viruses and plasmids by using crRNAs to guide the destroying of invading nucleic acids. Cas enzymes, which are CRISPR-associated enzymes, use bacteriophage DNA to continuously scan every DNA sequence inside the cell. If Cas enzymes find DNA that matches with the CRISPR sequences (as in case of viral infections), it initiates an immune response that terminates the viral DNA and protects bacteria from bacteriophage infection. The underlying mechanism behind the CRISPR system has many aspects; the mature crRNA base-paired to transactivating tracrRNA constitutes a two-RNA structure that guides the CRISPR-associated protein Cas9 to introduce double-stranded (ds) breaks in target DNA. On positions that are complementary to the crRNA-guide sequence, the complementary strand is cleaved by the Cas9 HNH nuclease domain. The Cas9 RuvC-like domain cleaves the noncomplementary strand. Doudna and Charpentier group in 2012 demonstrated that dual-tracrRNA:crRNA, when engineered as a single RNA chimera, also aims sequence-specific Cas9 dsDNA cleavage. This research group also found a family of endonucleases that uses dual-RNAs for site-specific DNA cleavage and high points the potential to exploit the system for RNA-programmable genome editing (Jinek et al., 2012).

6.6 Scientific advantage/applications The correctness and efficiency of this method of gene editing have revolutionized the creation of defined genetic and epigenetic variations in cells in culture and organisms from microbes, plants to animals. So far, ongoing uses include improving agricultural crops and livestock, developing new antimicrobial agents, and efforts to control disease-carrying insects with so-called gene drives (Wright et al., 2015). In human health, it has many aspects, and it is emerging as a basic research tool for use in human cells or embryos to help understand normal development, model human disease, and develop new treatments. Also, for gene editing in somatic cells, either ex vivo or in vivo, to treat or prevent disease. Gene editing may be done in gametes or embryos with the aim of correcting disease-causing mutations in the next generation (germline gene editing), although with serious ethical concerns in human cell research. The CRISPR/Cas system is extremely valuable as a gene-editing tool to do site-specific edits. Researchers can exploit CRISPR/Cas to target particular DNA sequences, such as the ones that cause fatal genetic diseases, and cleave the DNA at that specific target site. Scientists then use that cleavage to their advantage to have other proteins repair the DNA, replacing the pathogenic DNA with healthy DNA. CRISPR/Cas9 has allowed scientists to create genetically modified mice with significantly improved efficiency at a much faster pace (now its possible to obtain knock out animals in the first generation) (Teng et al., 2018). With the potential to knock out each of the genes in the genome, CRISPR knockout libraries can potentially be used to target regions of interest in the noncoding genome (e.g., promoters, enhancers), enabling screens not possible with RNA interference (Wang et al., 2014). One more promising usage of the CRISPR/Cas9 system is tagging genes with tag peptides or fluorescence markers. There are several tags that have been developed for the purpose such as HIS, FLAG, HA tags, and fluorescence proteins like RFP, EGFP, BFP, etc. to tag the gene of interest. This feature of fluorescence tagging proteins adds an enormous advantage in various areas like tracing subcellular location, real-time monitoring the expression, and dynamics of a protein in various conditions, which cannot be achieved by using conventional biochemistry or immunostaining assays. However, the production of such gene-tagged cell lines could be challenging (Xiang et al., 2019). Since the discovery of CRISPR/Cas system, there have been immense advances in the technological aspect that have allowed scientists to more precisely understand the host environment when studying viral pathogenesis in vitro, which includes CRISPR mediated gene editing, stem cell-derived organoid culture systems and induced pluripotent stem cells (iPSCs) (https://doi.org/10.3390/v11020124).

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Even before the discovery of the CRISPR/Cas system, scientists did have the tools to edit human genes. However, these available tools were very expensive and labor-intensive, and not that accurate as sometimes they did not work for particular DNA sequences. Since the CRISPR/Cas system originates from bacteria, it is less expensive and easier to generate the necessary constructs. Additionally, the CRISPR/Cas system can easily target any DNA sequence very precisely. It is much more specific, meaning that it could pinpoint and target the specific DNA sequence that researchers engineer it to target, and not accidentally target other DNA sequence (these are called “off-target” effects). Previous experiments done on modified zinc-finger TFs (Transcription Factors) have shown that viral replication and propagation may be inhibited by using synthetic transcriptional modulators (Papworth et al., 2003; Reynolds et al., 2003). It has also been very effective in restraining the expression of disease-associated genes, speeding up wound healing by inducing angiogenesis (Rebar et al., 2002). Also modified zinc-finger TFs (Transcription Factors), may be used for screening cellular targets for tracking the progression of disease like cancer and also to track drug resistance (Park et al., 2003). Encouraged by the success of modified zinc-finger transcription factors, scientists have paid more attention to TALENs and CRISPR-Cas9 technology to modify regulatory machinery, including transcriptional activators and repressors. For instance, TALENs and CRISPR-Cas9 have not only empowered speedy manufacture of customized genetic circuits (Gaber et al., 2014) but also opened up the possibility of modifying complex gene regulation networks (Nielsen and Voigt, 2004). Cellular reprogramming has made been made possible resulting in surprising results, which include differentiation of mouse embryonic fibroblast to skeletal myocytes (Chakraborty et al., 2014), Drosophila (Fruit fly) which has been a model of scientific experiments from ages, and dCas9 transcriptional effectors have even been used to efficiently mediate repression and activation of endogenous genes (Lin et al., 2015). Both TALEN and Cas9 activators have also been constructed to motivate transcription of latent HIV (Zhang et al., 2015), indicating their probable ability to work in conjugation with antiretroviral therapy for eliminating HIV infection. Due of the simplicity with which the CRISPR-Cas9 system can be used, compared to all other available gene-editing techniques, significantly, genome-wide screens using Cas9 transcriptional activators and repressors (Gilbert et al., 2014) can be easily applied in order to uncover genes involved in a number of diverse processes, which include but not limited to drug resistance and cancer progression. Specifically, CRISPR-based genome-scale screening methods have the potential to overcome many of the technical hurdles associated with other contemporary screening technologies, such as cDNA libraries and RNAi, indicating its potential for facilitating drug discovery and basic biological research.

6.7 Clinical aspect Precise genome manipulation is a powerful tool to treat or cure cancer, as well as many neurodegenerative diseases or HIV infection. There are several ongoing clinical trials worldwide involving CRISPR-Cas9 genome editing, which mostly involve ex vivo genome editing of human cells, during which cells are taken out from the body followed by CRISPR-based genome editing, and the edited cells are put back into the patient via autologous transplantation (Hoggatt, 2016). T cells are readily edited ex vivo and have the potential to fight infectious diseases like HIV. CRISPR-driven chimeric antigen receptor (CAR) T-cell therapy is the first US Food and Drug Administration approved commercial gene transfer therapy. Modified T cells are programmed to recognize cancerous cells through an engineered extracellular antigen-binding domain, which typically incorporates the variable region of an antibody with cancer specificity. Also, template-directed gene replacement via CRISPR-Cas9 can reverse the single point mutation causing Sickle Cell Disease in ex vivo hematopoietic stem/progenitor cells (HSPCs) at levels that are therapeutically viable (Dever et al., 2016; DeWitt et al., 2016). Widespread research work in mice and human cell lines has demonstrated that CRISPR can also be used to rectify mutations causing Duchenne muscular dystrophy, which recently progressed to restoring dystrophin protein expression in a canine model of the disease through systemic and intramuscular delivery of adeno-associated virus-encoded Cas9 (Amoasii et al., 2018). CRISPR-based treatments are also being developed for several brain disorders (Staahl et al., 2017) eye diseases such as congenital blindness, and diseases of the liver. Particular cells derived from an early embryo after fertilization but prior to the developmental stage are referred to as embryonic stem (ES) cells. Genome-editing methods have been extremely useful in generating an assortment of genetic modifications in human ES and iPS cells. Before the advancement of effective gene-editing tools, these cells had not shown promising results using conventional genetic modification tools like homologous recombination that had been used effectively in mouse ES cells. Using these implements in human cells ensued in very low frequencies of targeted recombination. However, the discovery of CRISPR/Cas9 documented significant improvements in efficiency. This enhancement enabled the rapid generation of tagged reporter cell lines, making it possible to follow differentiation pathways, look for interacting proteins, sort appropriate cell types, and investigate the functions of individual genes and pathways in cells, among many other

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applications (Hockemeyer and Jaenisch, 2016). Genome editing also permits the targeted rectification of disease mutations in patient-specific iPS cell lines to create genetically matched control lines. Such modified stem cell lines are used basically to conduct experimental studies, as well as preclinical investigations, to explore specific disease processes, and to test drugs that could be used to treat such diseases. Gene therapy in the field of medicine has got tremendous limelight, during which altered nucleic acid sequences are delivered into patient’s cells as a drug for the treatment of disease. This intervention is done to rectify defective genes so as to fight any disease condition. This technique could be done in somatic cells (Somatic gene therapy) and in egg or sperm cells (Germline gene therapy). CRISPR could have one more promising application in therapeutics of rare diseases. However, there are several bottlenecks, as for succeeding, a clearly defined clinical trial design is essential in order to deal with the several challenges in this area. Gene-editing techniques can provide answers to deal with the global challenges in treating diseases as pharmaceutical companies are embracing gene therapy advancement and as serviceable tools for the restoration of primary mutations separate the ability to create treatment by using expertise on particular disease mechanism. Since its first use in eukaryotic cells around 2013, the spread and enhancement, specifically of tools based on clustered regularly interspaced short palindromic repeats (CRISPR)/Cas prokaryotic RNA-guided nucleases has prompted a sweep of therapy-development studies for rare diseases (fdagov-public).

6.7.1 Limitations Despite many advantages of this system, there are some limitations to the current CRISPR/Cas9-based tools. Various research studies have investigated diverse aspects that affect the efficiency and specificity of the CRISPR-Cas9 system, such as Cas9 activity, target site selection and sgRNA design, delivery methods, off-target effects, and the incidence of homology-directed repair (HDR). Modern gene-editing techniques are quite precise, but they are not perfect yet. The procedure may be based on more trial and error, may go to the target cells but may also affect others. Even when CRISPRCas9 gets where it is needed, the edits can differ from cell to cell depending on the environmental conditions, for example, repairing two copies of a mutated gene in a single cell, but only one replica in another. For some genetic diseases, this may not matter, but it may have a significant impact if a single mutated gene sources the disorder. Another usual problem arises when edits are made at the undesirable location in the genome. There may be hundreds of these “off-target” edits that can be deleterious if they disturb healthy genes or crucial regulatory DNA. One significant limitation in biological studies and genetic therapies, is the off-target phenomena that generate undesired mutations at random sites, thus impacting precise gene modification. Lack of specificity in targeting, incomplete targeting, and so on could have devastating effects on patients undergoing gene therapy. Also, the size of the Cas9 protein is a constraint. The cDNA encoding S. pyogenes Cas9 is around 4.2 kb in size, so it is a bit larger than a TALEN monomer and much bigger than a ZFN monomer. As a result, it is challenging to deliver it via a viral vector (Laoharawee et al., 2018).

6.8 Ethical concerns Application of CRISPR/Cas9 involves risk, as it may produce off-target mutations that can be deleterious. Greater attention needs to be given so that benefits outnumber the risks involved. One problem is that large genomes may contain multiple DNA sequences that are identical or highly homologous to the intended target DNA sequence. CRISPR/Cas9 may cleave these unintended sequences causing mutations that may cause cell death or transformation. As gene drive continues operating in living systems, the possibility of off-target mutations may increase and pose considerable risk. Safety measures are necessary in order to avoid the dissemination of organisms that may cause ecological damage or affect human health. An additional issue is the regulation of patenting. Since long, transgenic organisms have been patented when they have industrial use; also, human gene sequences have been patented for clinical use, which led to the enormous growth of biotechnology. However, the practice of patenting may originate litigations. Already, there have been controversy and frictions among biotechnological companies over patenting CRISPR/Cas9 for therapeutic use in humans. There is always a possibility of genome editing in human germline; until now, all therapeutic interventions in humans using genome editing have been performed in somatic cells, but the experiment of Chinese researchers Liang and collaborators has created concern over the possibility of making changes in human germline (Liang et al., 2015), which will incorporate inheritable changes. In case of any undesirable mutation, it will be passed on to the next generation, which is definitely not desirable. The US National Institute of Health issued a statement, calling for a moratorium, banning NIH-funded research into genomic editing of human embryos (Wolinetz and Collins, 2019).

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The use of CRISPR/cas9 gene editing renews many social and ethical issues, not only with humans but also with other organisms and the environment. The risk assessment must be performed and safety issues must be considered in order to avoid ecological impairment.

6.9 Conclusion Various techniques of targeted gene editing hold incredible potential for providing therapeutic treatment to correct underlying genetic anomalies in order to cure several diseases. One of the most successful experimentations done so far is the usage of ZFN-mediated interruption of the HIV coreceptor CCR5, which was used to introduce HIV resistance into CD4þ T cells (Perez et al., 2008). Also, individuals diagnosed with HIV/AIDS have successfully been infused with gene-edited T cells and is in phase 1 clinical trial (Tebas et al., 2014). Apart from that facilitating the introduction of gene alteration that can enrich autologous cell therapies, targeted nucleases can also be combined with viral vectors, including AAV, to mediate genome editing in situ (Gaj et al., 2016). For instance, delivery of an AAV vector encoding a ZFN pair designed to target a defective copy of the factor IX gene, along with its repair template, led to efficient gene correction in mouse liver, increasing factor IX protein production in both neonatal and adult models of the disease (Li et al., 2011). Recently in vivo genome editing has enabled the rebuilding of dystrophin gene expression and the rescue of muscle function in mouse models of Duchenne muscular dystrophy (Long et al., 2015). Therapeutic gene editing in a mouse model of human hereditary tyrosinemia has also been reported using both hydrodynamic injections of plasmid DNA encoding CRISPR-Cas9 and by combining nanoparticle-mediated delivery of Cas9-encoding mRNA with AAV-mediated delivery of the DNA template for gene correction (Yin et al., 2014). More recently, a dual particle AAV system, where one AAV vector carried the Cas9 nuclease and a second carried the sgRNA and donor repair template, was able to facilitate correction of a disease-causing mutation in the ornithine transcarbamylase gene in the liver of a neonatal model of the disease (Yang et al., 2016). This work, in particular, showed that therapeutic levels of gene correction could be achieved in a regenerating tissue even when using multiple AAV particles. Although highly promising, numerous hurdles still need to be overcome for in vivo applications of genome editing to reach its full potential. One of the obstacles is to develop methods that can facilitate nuclease delivery or expression to only diseased cells or tissues, and developing new strategies that can enhance HDR in disease-associated postmitotic cells in vivo. Despite the successes already achieved, many challenges remain before the full potential of genome editing can be realized. First and foremost is the development of new tools capable of introducing genomic modifications in the absence of DNA breaks. Targeted recombinases, which can be engineered to recognize specific DNA sequences and even integrate therapeutic factors into the human genome, are one such option (Gaj et al., 2013). More recent work has indicated that singlebase editing without DNA breaks can be achieved using an engineered Cas9 nickase complex (Komor et al., 2016), although it remains unknown how effective this technology is in therapeutically relevant settings. By linking genomic modifications induced by targeted nucleases to their own self-degradation, self-inactivating vectors are also poised to improve the specificity of genome editing, especially because the frequency of off-target modifications can be directly proportional to the duration of cellular exposure to a nuclease (Pruett-Miller et al., 2009). In addition, much of the knowledge behind genome engineering has been obtained in immortalized cell lines. However, in the case of regenerative medicine, it is highly desirable to genetically manipulate progenitor or stem-cell populations, both of which can differ markedly from transformed cell lines with respect to their epigenome or three-dimensional organization of their genomic DNA. These differences can have profound effects on the ability of genome-editing tools to either modify a specific sequence or regulate gene expression. Although the union between genome engineering and regenerative medicine is still in its infancy, realizing the full potential of these technologies in stem/progenitor cells requires that their functional landscape be fully explored in these genetic backgrounds. Only then will genome-editing technologies truly be able to reprogram cell fate and behavior for the next generation of advances in synthetic biology and gene therapy. There are still some fundamental questions remaining concerning the use and optimization of genome-editing methods both in cultured cells and in experimental multicellular organisms (e.g., mouse, insects, plants). Such basic discovery research is essential for improving any future applications of genome editing. Applications of genome editing in laboratory research also have added powerful new tools that are contributing greatly to the understanding of basic cellular functions, metabolic processes, immunity and resistance to pathological infections, and diseases such as cancer and cardiovascular disease.

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Genome editing is a powerful new tool for making precise alterations to an organism’s genetic material. Recent scientific advances have made genome editing more efficient, accurate, and flexible than ever before. These advances have encouraged scientists from around the globe in every possible way in which genome editing can improve human health.

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Laoharawee, K., DeKelver, R.C., Podetz-Pedersen, K.M., Rohde, M., Sproul, S., Nguyen, H.O., Nguyen, T., St Martin, S.J., Ou, L., Tom, S., Radeke, R., Meyer, K.E., Holmes, M.C., Whitley, C.B., Wechsler, T., McIvor, R.S., 2018. Dose-dependent prevention of metabolic and neurologic disease in murine MPS II by ZFN-mediated in vivo genome editing. Mol. Ther. 26 (4), 1127e1136. Li, H., Haurigot, V., Doyon, Y., Li, T., Wong, S.Y., Bhagwat, A.S., Malani, N., Anguela, X.M., Sharma, R., Ivanciu, L., Murphy, S.L., Finn, J.D., Khazi, F.R., Zhou, S., Paschon, D.E., Rebar, E.J., Bushman, F.D., Gregory, P.D., Holmes, M.C., High, K.A., 2011. In vivo genome editing restores haemostasis in a mouse model of haemophilia. Nature 475 (7355), 217e221. Liang, P., Xu, Y., Zhang, X., Ding, C., Huang, R., Zhang, Z., Lv, J., Xie, X., Chen, Y., Li, Y., Sun, Y., Bai, Y., Songyang, Z., Ma, W., Zhou, C., Huang, J., 2015. CRISPR/Cas9-mediated gene editing in human tripronuclear zygotes. Protein Cell 6 (5), 363e372. 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Mali, P., Yang, L., Esvelt, K.M., Aach, J., Guell, M., DiCarlo, J.E., Norville, J.E., Church, G.M., 2013. RNA-guided human genome engineering via Cas9. Science 339 (6121), 823e826. Nielsen, A.A.K., Voigt, C.A., 2004. Multi input CRISPR/Cas genetic circuits that interface host regulatory networks. Mol. Syst. Biol. 10 (11), 763e763. Nuñez, J.K., Lee, A.S.Y., Engelman, A., Doudna, J.A., 2015. Integrase-mediated spacer acquisition during CRISPReCas adaptive immunity. Nature 519 (7542), 193e198. Papworth, M., Moore, M., Isalan, M., Minczuk, M., Choo, Y., Klug, A., 2003. Inhibition of herpes simplex virus 1 gene expression by designer zinc-finger transcription factors. Proc. Nat. Acad. Sci. U.S.A. 100 (4), 1621e1626. Park, C.Y., Kim, J., Kweon, J., Son, J.S., Lee, J.S., Yoo, J.E., Cho, S.R., Kim, J.H., Kim, J.S., Kim, D.W., 2014. Targeted inversion and reversion of the blood coagulation factor 8 gene in human iPS cells using TALENs. Proc. Nat. Acad. Sci. U.S.A. 111 (25), 9253e9258. 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Chapter 7

RNAi for livestock improvement Uzma Noor Shah1, Shanmugapriya Gnanasekaran2, Sukanta Mondal1, 3, I.J. Reddy1, S. Nandi1, P.S.P. Gupta1 and D.N. Das2 1

ICAR-National Institute of Animal Nutrition and Physiology, Bangalore, Karnataka, India; 2Genetics Laboratory, Dairy Production Section, ICAR-National Dairy Research Institute (SRS), Southern Regional Station, Bangalore, Karnataka, India; 3Principal Scientist, ICAR-National Institute of Animal Nutrition and Physiology, Adugodi, Bangalore, Karnataka, India

7.1 Introduction RNA interference is a regulatory system controlling the activity of genes present in eukaryotic cells (i.e., cells having a defined nucleus). RNAi function is to silence/deactivate genes specifically. In modern biology, RNAi brought the most significant scientific breakthrough by letting us observe the outcome of the loss of function of specific genes in mammalian systems directly. Initially, RNAi technique was used by a few laboratories, but at present, it has become essential for gene function studies. It has wide applications in functional genomics like in vivo knockdown, protein knockdown studies, phenotype analysis, function recovery, pathway analysis, drug target discovery, and many more. RNAi is naturally in-built and conserved self-defensive mechanism initiated by double-stranded RNA (dsRNA) that confines the level of transcription by two methods: either by suppressing transcription or by activating sequence-specific degradation of RNA (Singh et al., 2019). It protects the host from endogenous, as well as exogenous invading nucleic acids simply by regulating the gene expression. Living organisms have evolved numerous mechanisms in order to protect themselves against invading pathogens. Few of the mechanisms comprise of restriction digestion of the DNA of infectious agents by restriction endonucleases, modification, and editing of pathogen’s genome by clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) (CRISPR/Cas-9) system, and gene silencing by double-stranded RNA, etc.

7.2 History behind RNAi Scientists, at the beginning of the 1990s, first noticed that RNA inhibited protein expression in plants and fungi. The phenomenon of RNAi, i.e., ability to silence genes, was discovered in the 1990s by two American scientists Andrew Z. Fire and Craig C. Mello, who shared the Nobel Prize for Physiology or Medicine in 2006 for their work. In the year 1998, Fire and Mello, while working on the worm Caenorhabditis elegans, discovered that the actual source of the inhibition was double-stranded RNA, and hence, they called this phenomenon as RNA interference (RNAi). RNA interference (RNAi) is a naturally evolved and a conserved self-defensive mechanism triggered by double-stranded RNA (dsRNA) that limits the transcript levels either by suppressing transcription or activating sequence-specific degradation of RNA. Although research in C. elegans was encouraging, the use of RNAi was restricted to lower organisms because delivering of long dsRNA for RNAi to happen was inhibitory in mammalian cells nonspecifically. Then in 2001, it was shown that shorter RNAs (siRNA) were able to trigger RNAi in mammalian cells (Elbashir et al., 2001) directly without producing any nonspecific effects as found with longer dsRNAs. In 2006, only 8 years after the discovery of siRNA, the Nobel Prize in Physiology or Medicine was awarded to Fire and Mello. During the research by Fire and Mello on gene expression studies in C. elegans, they found that by injecting mRNA (sense strand) that encodes for muscle protein production, no response was elicited from the worms. After that, they also injected antisense RNA that can pair with the sense sequence mRNA, which also failed to elicit any response from the worms. Finally, when they injected both the sense and the antisense RNA together, they observed some twitching movements from the worms. The result was surprising because they know that the same kinds of movements were observed from worms whose genes encoding for muscle protein were rendered dysfunctional. In order to explain their results, Fire and Mello hypothesized that the dsRNA that is formed by the binding of the sense and antisense RNA is able to silence the gene carrying just the same code as the RNA molecule. In order to validate their

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hypothesis, they injected dsRNA that codes for specific proteins and observed that the genes carrying exactly the same code as the RNA that was injected, were silenced. RNA interference discovery is significant for two reasons: 1. Scientists can knockdown the production of any protein in a cell specifically with RNAi technology. 2. Earlier scientists were assuming introns as just junk DNA and that they have very little purpose, buy now they know that many of these introns code for RNAi elements.

7.3 Versatility of RNA molecule RNA is a very versatile nucleic acid both in its abundance, as well as in its functions. RNAi has a wide range of applications like enhancing or reducing gene expression through numerous mechanisms. It may be noted down that gene suppression is triggered by RNA interference (RNAi), microRNA (miRNA), small interfering RNA (siRNA), antisense RNA, and synthetic hairpin RNA (shRNA), etc. Protein synthesis is affected by messenger RNA (mRNA) present in the cells. This mechanism is similar to gene therapy wherein the genes incorporated into cells produce desirable proteins for curing genetic disorders, etc. or produce some favorable effects (Balmayor and Evans, 2019). miRNAs and short siRNAs are of utmost biomedical implication in gene-regulation mechanisms in plant and animal organisms (Carrington and Ambros, 2003). RNAi works by limiting the levels of transcription either by suppressing transcription (transcription gene silencing or TGS) or by activating RNA degradation in a sequence-specific manner (posttranscriptional gene silencing or PTGS) (Agrawal et al., 2003). RNAi was discovered in plants initially, but later on found in many different organisms like nematodes, arthropods (insects and ticks), protozoa mammals, and even cell lines (Agrawal et al., 2003). The first evidence for RNAi came from C. elegans, followed by subsequent works in Drosophila melanogaster, and mammalian cells (Elbashir et al., 2001). RNAi has a very important role in regulating genes and also in triggering cellular defense mechanism against infection by RNA viruses like influenza viruses and rhabdoviruses, a group that is the causative agent of rabies. Many plants and animals have developed antiviral RNAi genes that encode small segments of RNA molecules having complementary sequences to viral sequences. This complementarity of interfering RNA with the viral sequence enables it to bind and inactivate specific RNA viruses. RNAi is considered as an innate defensive mechanism by which cells are able to suppress the activity of transposons (jumping genes).

7.4 Mechanism of RNAi In order to silence gene expression, RNAi is an effective way that requires a few molecules of dsRNA/cell. dsRNA comes from viruses and transposon activities, and under natural conditions, it can be introduced into cells experimentally. RNAi is achieved by producing siRNAs derived from viruses and when introduced into cells bind with just the right complementarity to target viral sequence (Fay and Langlois, 2018). It is also a novel inbuilt regulatory mechanism that limits the endogenous protein levels either by degrading the mRNA or by inhibiting translation.

7.5 Pathways of RNA silencing RNA silencing can be classified into two major categories; posttranscriptional gene silencing (PTGS) and transcriptional gene silencing (TGS).

7.5.1 Posttranscriptional gene silencing (PTGS) The posttranscriptional gene silencing pathway is triggered by the production of dsRNA. The potential inducers of PTGS include RNA and DNA viruses, transgenic sense RNA, antisense RNA, inverted repeats, or aberrant RNAs (RNA molecules lacking polyA tail or 50 cap (Fig. 7.1). The dsRNA trigger is processed by an RNase III-like dicer protein (DCL) into characteristic 20e25 nucleotide (nt) long, small-interfering RNA (siRNA) with 2-nt 30 overhangs (Dunoyer and Voinnet 2005). DCL1 is involved in the biogenesis of miRNAs (Bartel, 2004), whereas DCL2, DCL3, and DCL4 generate siRNAs of 22, 24, and 21-nt sizes, respectively (Dunoyer and Voinnet 2005; Xie et al., 2005). The 30 -terminal nucleotide of siRNA is methylated by the methyltransferase HEN1 for their protection from degradation and polyuridylation (Boutet et al., 2003; Chen, 2005). Host RNA-dependent RNA polymerase (RdRp) and several other cellular proteins are involved in the siRNA amplification process that helps in the systemic spread of RNA silencing (Baulcombe, 2004). The siRNAs are denatured and recruited into a multiprotein RNA-induced silencing complex (RISC). RISC is then guided to the target mRNA by the complementary 21-nt siRNA. Argonaute (AGO) cleaves the mRNA target (Lima et al., 2016). PTGS is often referred to as the “cytoplasmic RNA silencing pathway.”

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PTGS pathway

TGS pathway

Transgenic sense RNA Transgenic antisense RNA Viral RNA Direct repeat (DR) RNA

Heterochromatin Repeated elements: IR, DR DNA virus Transposon

Over abundant RNA

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dsRNA biosynthesis Inverted-repeat (IR) RNA

dsRNA biosynthesis

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dsRNA processing DCL4

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AGO4 RITS – RNA Induced TranscriptionalSilencing Complex

FIGURE 7.1 RNA interference by posttranscriptional gene silencing (PTGS) and transcriptional gene silencing (TGS).

7.5.2 Transcriptional gene silencing (TGS) Transcriptional gene silencing shares similar components and uses a similar siRNA-guided process as PTGS. TGS takes place inside the nucleus, and its role is to initiate and maintain the heterochromatic state, in certain regions of the genome (Fig. 7.1). TGS targets homologous DNA sequences and causes DNA methylation that suppresses transcription (Vaucheret and Fagard, 2001). The dsRNA triggers are synthesized by RdRp2, diced into 24-nt RNA by DCL3 in Cajal body-like subdomains within the nucleus. The 20 -OH is methylated by dsRNA methyltransferase HEN1. Following the degradation of the passenger strand, the guide strand binds to AGO4, or in some cases to AGO6 and AGO9, depending upon the loci and tissue. The complex is then associated with chromoproteins, histone H3K9 methylase and DNA-methylating enzymes (DRM1, DRM2), PolIVb and chromomethylase (CMT3) to form RNA-induced transcriptional silencing (RITS) complex, which is involved in the maintenance of histone and DNA cytosine methylation (Matzke et al., 2009). Mammalian cells probably lack the siRNA amplification mechanisms that confer RNAi potency and longevity in organisms such as worms or plants. There is a range of applications of RNAi, such as suppressing or enhancing the expression of genes through diverse mechanisms. Gene suppression is mediated through RNA interference (RNAi), microRNA (miRNA), small interfering RNA (siRNA), antisense RNA, and synthetic hairpin RNA (shRNA).

7.6 The miRNA pathway RNAi also takes place in the endogenous gene silencing machinery using microRNAs (miRNAs). miRNAs are a class of short regulatory RNAs of 16e28 nt in length in humans and play critical roles in cell proliferation, differentiation, and metabolism. It was reported that 16e28 nt small RNAs mainly include miRNAs. The pathways for miRNAs biogenesis are complex and include posttranscriptional modifications. In this respect, investigation of modifications on 16e28 nt small RNAs can promote our understanding of the regulatory roles of noncoding RNAs. Primary microRNAs (pri-miRNAs) are

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first processed to form pre-miRNAs by the enzyme Drosha, and pre-miRNAs then enter the RNAi pathway. These transcripts are generated by PolII, termed as pri-miRNA, and possess 50 cap and 30 polyA tail hallmarks (Kim, 2005). From this transcript, arises the 70e100 nt long pre-miRNA. The pre-miRNA is transported to the cytoplasm via exportin-5 (Lund et al., 2004). It is then digested by a double-strand specific RNase III enzyme called Dicer1 (DCL1) in conjunction with HYL1 (Tang et al., 2003). The resultant 19e24 nt miRNA, methylated by HYL1, is bound by a complex that is similar to the RNA-induced silencing complex (RISC) that participates in cleaving the target mRNA or repressing the translation of the target transcript resulting in the reduced accumulation of the corresponding protein. The miRNA: miRNA duplex is transported to the cytoplasm by Exp-5 homolog, Hasty (Bollman et al., 2003). The first miRNA, lin-4, was discovered in C. elegans and was found to control the timing of various stages of larval development by blocking translation of the protein LIN-14 (Lee et al., 1993). Since then, many more miRNAs have been discovered in invertebrates and mammals, and these have been shown to be critical during developmental timing, cell proliferation, differentiation, and apoptosis, and signaling pathways. The miRNAs, while allowing mismatches, can either repress the target mRNA translation (mostly in mammals) or facilitate mRNA destruction (mostly in plants). Multiple miRNAs have been characterized for their physiological roles in cancer and other diseases. Lackinger et al. (2018) demonstrate that a large cluster of miRNAs, which emerged in placental mammals, functions as a repressor of social behavior. This discovery has significant implications for our understanding of the brain, behavior, and particularly psychiatric syndromes, which have been shown to display alterations of these molecules (Murray, 2019).

7.7 piRNA piRNAs have also been found to be expressed in the germline of many other metazoan species, including mouse, rat, zebrafish, Xenopus, silkworm, and C. elegans. There are hundreds of thousands, if not millions, of distinct piRNA sequences within a species, and piRNA sequences are not conserved among different species. The piRNAs (w25e30 nt) are different in size from miRNAs and siRNAs (21e24 nt). piRNAs are processed from long, single-stranded precursor transcripts. In addition, the 50 ends of piRNAs have a preference for uridine. The 30 ends of piRNAs are 20 -O-methylated. By definition, piRNAs are bound to PIWI proteins. Two classes of piRNAs are generated during mouse spermatogenesis: prepachytene and pachytene piRNAs.

7.8 Transgenesis in livestock improvement Initially, it was demonstrated that a single gene (human growth hormone) transferred into a one-cell mouse embryo could result in a dramatic change in the phenotype (50% increase in body size and weight) of the resulting mouse (Palmiter et al., 1982). The first transgenic livestock was produced more than 30 years ago by pronuclear microinjection (Hammer et al., 1985). A wide range of genetically modified large animals has been produced by this technology for applications in agriculture and biomedicine, with the use of transgenic livestock for “gene pharming” already at commercial level (Kues and Niemann, 2004; Niemann and Kues, 2007). Although pronuclear microinjection is relatively straight-forward, it has several major disadvantages, including low efficiency, random integration, and variable expression patterns. Studies were focused on the development of substitute technologies for the improvement the efficiency and decreasing the cost of generating transgenic livestock. These include sperm mediated gene transfer (Lavitrano et al., 1989, 2002; Chang et al., 2002), intracytoplasmic injection (ICSI) of sperm heads carrying foreign DNA (Perry et al., 1999, 2001), injection of oocytes and/or embryos by different types of viral vectors (Hofmann et al., 2004), RNA interference technology (RNAi) (Clark and Whitelaw, 2003), somatic cell nuclear transfer (SCNT) (Schnieke et al., 1997; Cibelli et al., 1998; Baguisi et al., 1999; Dai et al., 2002; Lai et al., 2002). Transgenic applications will become more extensive when technologies for precise genetic engineering are fully developed.

7.9 RNAi in livestock The identification and characterization of the process of RNA interference in Caenorhabditis (Rocheleau et al. 1997; Fire et al. 1998) allowed for the development of RNAi-based technology for use in cells and ultimately in transgenic livestock (Golding et al., 2006; Jabed et al., 2012). RNAi has been employed in mammalian cells successfully. Many different methods have been employed for the siRNA knock down of specific genes in mammalian cells. RNAi has been utilized for transient, as well as stable silencing of genes in mammalian cells. Tuschl and colleagues demonstrated that RNA interference could be directly mediated by small interference RNA (siRNA) in cultured mammalian cells. However, because

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siRNA does not integrate into the genome, the RNAi response from siRNA is only transient. In order to induce stable gene suppression in mammalian cells, Hannon and his colleagues utilized RNA Pol III promoter-driven (e.g., U6 or H1) expression of short hairpin RNAs (shRNAs). In order to specifically control/suppress the translation of mRNAs from either endogenous or exogenous viral elements, RNA interference is involved in gene regulation and also for therapeutic uses (Dallas and Vlassow, 2006). For a transient gene silencing, synthetic siRNAs are transfected into either cells or early embryos (Clark and Whitelaw, 2003; Iqbal et al., 2007). For stable gene silencing, the siRNA sequences should be incorporated into a gene construct. The lentiviral vector technology, along with siRNA, is an extremely efficient tool. RNAi silencing of PERV has been done successfully in porcine primary cells (Dieckhoff et al., 2007), as well as the silencing of the prion protein gene (PRNP) in cattle embryos (Golding et al., 2006). Germline transmission of lentiviral siRNA has been done in rats over three generations (Tenenhaus Dann et al., 2006). In the absence of being able to make knockouts in livestock, attempts were made to downregulate gene expression using antisense RNA constructs (Sokol and Murray, 1996). Antisense RNA-based techniques to downregulate gene expression in transgenic animals, but with limited success (Sokol and Murray, 1996). shRNAs for RNAi expressing short hairpin RNA (shRNA) from RNA polymerase III promoters in plasmid or viral-based vectors is an efficient way of silencing target genes. shRNAs are produced as single-stranded 50e70 nucleotides molecules that form stem-loop structures. The shRNA mimics the endogenous microRNA (miRNA) pathway to trigger the cleavage of shRNAs, generating siRNA for the silencing of specific genes (Brummelkamp et al., 2002; Paddison et al., 2002). The shRNA encoding DNA fragments can be made by chemically synthesizing 50e70 nt long oligonucleotides that can be annealed and cloned into a vector. The shRNA-based transgene strategy has substantial benefits than vaccination by offering potential subserotype protection when using multiple-shRNA expression systems targeting different viruses. Zinc finger nucleases (Shi and Berg, 1995) and with their development the potential to directly target a sequence for mutation, KO, or transgene insertion in a specific location in the genome became feasible (Durai et al.2005). The first livestock produced using ZFNs were rabbits (Flisikowska et al., 2011) and pigs (Whyte et al., 2011). The advent of ZFNs designed to cut at specific sites in genomic DNA revealed the potential of this approach for genetic engineering and gene editing (Jabalameli et al., 2015). However, ZFNs, as class of designer endonucleases, are relatively cumbersome to design and produce, although they are efficient to use. The realm of designer nucleases was rapidly expanded, first by the discovery of the code for DNA binding specificity for the bacterial transcription activator-like effector nucleases (TALENs) and more recently by the discovery and manipulation of the clustered regularly interspaced short palindromic repeats(CRISPR) and the CRISPRassociated endonucleasecas9 (CRISPR/Cas9) system (Jinek et al., 2012). CRISPR RNAs (crRNA) and CRISPR-associated proteins (Cas) are part of an adaptive immune response in bacteria, as well as in archaea, and protects them against viral infections. The CRISPR can be found on both chromosomal and plasmid DNA. When the same virus or plasmid invades again, the corresponding invading DNA will act as a memory, recognize, interfere, and provide immunity. The CRISPR system identifies, cuts, and destroys foreign DNA. CRISPR/Cas9 consists of two parts: single-guide RNA (sgRNA) and Cas9 endonuclease. The two components form a complex to cleavage target DNA sites; sgRNA is derived from the fusion of tracrRNA (trans-activating CRISPR RNA) and crRNA (CRISPR RNA). This complex is known as “guide RNA (gRNA),” which then guides a protein called Cas9 to recognize viral DNA. Cas9 is an endonuclease that produces double-strand breaks (DSB) in a genome.

7.10 Transgenic expression of RNAi-inducing molecules The most common method for the delivery of RNAi-inducing molecules till date has been via genomic integration of shRNA constructs (Gama Sosa et al., 2010; Maksimenko et al., 2013). Conventional methods of transgene insertion include the following: (i) DNA microinjection Pronuclear injection, a technique developed in the mouse, which involves the direct introduction of a DNA construct into one of the two pronuclei of the fertilized egg, was the technique used to produce the early transgenic livestock. The transgene with shRNA is introduced into the nucleus of a fertilized egg where it is arbitrarily inserted into the host genome (Fig. 7.2). The fertilized egg is then transferred to a foster mother; after the successful integration, the offspring will carry the transgene (Gordon and Ruddle, 1981). More recently, nuclear transfer techniques have been adapted to allow more precise modifications of the genome, such as the disruption of specific endogenous genes.

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FIGURE 7.2 Landmark events in transgenic livestock research.

(ii) Embryonic stem cell-mediated gene transfer Embryonic stem cells resulting from early-stage mouse embryos can be cultured indefinitely. The targeting vector contains the endogenous gene carrying the required shRNA and is incorporated into these cells and inserted into the genome by homologous recombination. The altered embryonic stem cells produced are then injected into early mouse embryos. If the altered cells donate to the germ cells of the mouse, progeny in a subsequent mating will inherit the shRNA expressing gene (Smithies et al., 1985). (iii) Somatic cell nuclear transfer Somatic cells are maintained in culture. By means of homologous recombination, a target vector containing an endogenous gene carrying the required shRNA is introduced into these somatic cells and inserted into the genome. Then the somatic cells are screened to find out the cells that have properly integrated the shRNA. Oocytes are then enucleated, and nuclei of shRNA-integrated cells are transferred into the enucleated oocytes, and fused with an electrical pulse. After being fused, the cells behave like in vitro fertilized embryos, and are grown in culture till the blastocyst stage, after which they are transferred into recipient animals. (iv) Retrovirus-mediated gene transfer Retroviruses have the capability to infect host cells and integrate into their genome. Hence, they are most commonly used as vectors for transgenic applications, creating a chimeric embryo with the DNA inserted randomly (Nagano et al., 2001). (v) Transposon-mediated gene transfer Transposons or Jumping genes are mobile genetic elements that can facilitate the transposition of DNA from plasmid vectors into chromosomes. During transposition, the enzyme transposase recognizes transposon-specific inverted terminal repeat sequences flanking the shRNA transgene, and moves the shRNA transgene into a random chromosomal site (Macdonald et al., 2012). (vi) Sperm-mediated gene transfer shRNA with the transgene is inserted into the sperm head and then used to fertilize eggs (Moreira et al., 2007). DNA can be introduced to the sperm head in various ways, including transfection (Fig. 7.2) (Collares et al., 2011), attachment by antibodies (Chang et al., 2002) and disruption by repeated freezeethaw cycles or also by exposure to detergents (Perry et al., 2001).

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(vii) Lentivirus mediated transgenesis Lentiviruses are proven to be efficient vectors for delivering genes into oocytes and zygotes. For instance, transgenic cattle have been generated by injecting lentiviral vectors into the perivitelline space of oocytes (Hofmann et al., 2004) and transgenic pigs have been produced by injecting lentiviruses into the perivitelline space of zygotes resulting in a high proportion of transgene expressing founder animals from which several lines of transgenic pigs were generated (Hofmann et al., 2003; Whitelaw et al., 2004). Lentiviral vectors, in combination with RNA interference, efficiently deliver interfering RNAs that can block or destroy the viral DNA of invading viruses to generate transgenic animals with resistance to viral diseases (Fig. 7.2). Although RNA interference is very versatile and can also be combined with other transgenesis methods, embryo mediated and cell mediated, to deliver an expression construct for a small interfering RNA, it can be applied for the purpose of precisely knocking down the expression of particular endogenous genes ((Jabed et al., 2012). Lentivirusmediated transgenesis in livestock has generated extraordinary high yields of transgenic animals because of multiple integration events. Regrettably, multiple integrations have the shortcoming of an increased probability of unwanted side effects caused by either oncogene activation or insertional mutagenesis. Other problems are gene silencing by DNA methylation due to the presence of viral sequences (Hofmann et al., 2006) and a high frequency of mosaicism in founder animals.

7.11 Applications of RNAi in livestock 1. Combating contagious/infectious diseases: Gene knockdown could also be applied for suppressing of infectious pathogens, mostly viruses, by targeting the RNA of the invading agent. Infectious diseases affect livestock production adversely, as well as animal welfare, and have direct impacts upon human health and public opinion of livestock production. Using transgenic animals for the benefit of animal/ human health is becoming progressively more due to the combination of new technologies that facilitate the efficient production of genetically modified animals, exciting potent tools to modify gene expression, and advancement of our basic understanding of causative agents and the mechanism of disease. Infectious diseases are the main problems in animal agriculture, with overlapping issues of intensive, as well as extensive production systems (Perry et al., 2013). There is no doubt regarding advancement in research methodologies, diagnostic facilities, and manufacturing methods, but there are many infectious diseases for which no effective vaccines exist. Additionally, vaccination can bring about the emergence of resistant and more infectious viruses, e.g., Mareks disease virus of poultry (Witter, 1997; Nair, 2005). Many viral vaccines available today are not effective against the existing strains in the field, so new vaccines have to be generated from field strains with each new outbreak, which can take months or so. Other significant concerns include global availability, field compliance, effectiveness, and safety, etc. The development of vaccines is becoming more complex and expensive. There is also significant concern by people about antibiotic use and other pharmaceutical products in agricultural animals, which has a significant impact on the control of many diseases. Current work is exploring different uses of RNAi to overcome these challenges to combat infectious diseases in livestock. 2. Direct targeting of pathogens RNAi has an advantage over conventional vaccines in that it is highly specific for the pathogen and can be made to a large scale very quickly (Dykxhoorn and Lieberman, 2005). Entire genomes of emerging viruses can be sequenced within a day with the advent of high throughput sequencing. Once this is known, siRNA can be rapidly designed using published algorithms and can be tested in vitro and in vivo within days. By comparison, it can take months to screen the vast number of candidate small drug molecules against a new virus or to develop and test inactivated vaccines. RNAi has been shown to inhibit a wide range of agriculturally significant pathogens both in vitro and in vivo, including foot and mouth disease virus (FMDV; Jiao et al., 2013; Gismondi et al., 2014), African swine fever virus (Keita et al., 2010), classical swine fever virus (Porntrakulpipat et al., 2010), influenza A (Stoppani et al., 2015), highly pathogenic avian influenza (Stewart et al., 2011), chicken anemia virus (Hinton and Doran, 2008), bovine viral diarrhea virus (Lambeth et al., 2007), and infectious bursal disease virus (Wang et al., 2010). When directly targeting a pathogen, the RNAi target site needs to be in a highly conserved region of the genome (usually within a gene essential for replication) and must take into account the 3-D structure of the viral genome. In the case of FMDV, the conformational folding of the RNA genome has been shown to inhibit the activity of a range of siRNA sequences (Gismondi et al., 2014).

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Many examples of transgenic animals expressing shRNA targeting disease agents have been published so far. The transgenic goat that expressed shRNA, targeting the prion protein that causes contagious spongiform encephalopathies, was the first report (Golding et al., 2006). The transgenic fetus was collected on the 81st day of the gestation period and had a reduction of >90% in prion protein abundance in the brain as compared to a nontransgenic fetus at the same stage of development. A similar approach was used to produce a viable calf expressing an antiprion shRNA (Wongsrikeao et al., 2011), reported that the prion mRNA and protein levels in nervous tissue were 24% and 86% lower, respectively as compared to the control animals. Recently porcine reproductive and respiratory syndrome shRNA pigs were produced. These pigs survived 11 days longer than the control pigs when challenged with the porcine reproductive and respiratory syndrome (Li et al., 2014). At the same time, transgenic mice expressing two anti-FMDV shRNA, targeting the viral polymerase protein 3D and the 2B regions of the nonstructural protein, showed 19%e27% higher survival rates as compared to the wild type mice (Chang et al., 2014). In all of the above examples, a moderate knockdown of the disease of the gene target was observed. RNAi, in combination with transgenic technology, offers the great possibility to genetically engineer livestock to promote resistance to viral infections and prion diseases. There are numerous examples of exogenously delivered siRNA inhibiting viral infections that include some important human pathogens like the influenza virus, respiratory syncytial virus, and hepatitis viruses B and C in animal models. siRNA therapeutics targeting respiratory syncytial virus and Ebola virus have been evaluated in clinical trials (Kanasty et al., 2013). Inhibition of influenza virus infection has been demonstrated in mice and chicken embryos (Hinton et al., 2014), and adenovirus constructs expressing shRNA inhibited FMDV in guinea-pigs successfully (Xu et al., 2012). Exogenously delivered siRNA is becoming a useful therapeutic tool and is being considered as an alternative strategy in the control of livestock diseases. RNAi approaches are being used to amplify the vaccine response. For example, the siRNA knockdown of suppressor of cytokine signaling one in dendritic cells before exposure to HIV proteins led to increased production of antibody and expansion of antigen-specific T-cell populations in mice (Song et al., 2006). In vivo studies done using IL-10-silencing siRNA, codelivered with plasmid DNA encoding for the hepatitis-B surface antigen, showed significant “switching” toward a T helper one immune response and increased the cytotoxic T-cell response (Singh et al., 2008). Transgenic pigs bearing an hMT-pGH construct (human metallothionein promoter driving the porcine growth hormone gene) showed significant improvement in economically important traits, including growth rate, feed conversion, and body composition (muscle/fat ratio) without the pathological phenotype seen with earlier GH constructs (Pursel et al., 1989; Nottle et al., 1999). Similarly, transgenic pigs carrying the human insulin-like growthfactor-I gene (hIGF-I) had w30% larger loin mass, w10% more carcass lean tissue, and w20% less total carcass fat (Pursel et al., 1999). 3. Wool Production It was observed that transgenic sheep having a keratin-IGF-I construct showed expression in the skin, and the amount of clear fleece was about 6.2% greater in transgenic than in nontransgenic animals (Singhal and Kansara, 2010). No adverse effects on health or reproduction were noticed. Only limited success was achieved in approaches designed to alter wool production by transgenic modification of the cysteine pathway although cysteine is known to be the rate-limiting biochemical factor for wool production (Kassim et al., 2019). 4. Pharmaceutical Production Gene “pharming” can be defined as the production of recombinant human proteins in the mammary gland of transgenic animals. This technology overcomes the limitations of traditional and recombinant DNA technology (Meade et al., 1999; Rudolph 1999) and has progressed to the phase of commercial application (Ziomek, 1998; Dyck et al., 2003). The mammary gland is the preferred site for production mainly because of the quantities of protein that can be produced in the mammary gland using mammary gland-specific promoter elements and established methods for extraction/purification of the respective protein (Meade et al., 1999; Rudolph, 1999). Guidelines developed by the Food and Drug Administration (FDA) require monitoring the animals’ health in a specific pathogen-free facility, sequence validation of the gene construct, characterization of the isolated recombinant protein, and monitoring the genetic stability of the transgenic animals over numerous generations. This has necessitated the use of animals from scrapie free countries (New Zealand) and maintenance of production animals under highly strict hygienic conditions. Several products derived from the mammary glands of transgenic sheep and goats have progressed to advanced clinical trials (Echelard et al., 2006). Phase III trials for antithrombin III (ATIII) (ATryn from GTC-Biotherapeutics, USA), produced in the mammary gland of transgenic goats, have been successfully completed and the recombinant product was approved as a drug by the European Medicines Agency (EMEA) in August 2006. This protein is the first product from a transgenic farm animal to be accepted as a fully registered drug. ATryn is registered for the treatment of heparin-resistant patients undergoing cardiopulmonary bypass surgeries. GTC-Biotherapeutics has also expressed not less than 11 other transgenic proteins in the mammary gland of transgenic goats. The enzyme a-glucosidase from the milk of transgenic rabbits has been given drug status and has been successfully used for the treatment of Pompe’s disease (van den Hout et al., 2001).

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An extremely interesting new development is the production of recombinant proteins in the mammary gland of transgenic animals for use as antidotes against organophosphorus compounds used as insecticides in agriculture and chemical warfare. Butyrylcholinesterase is a potent prophylactic agent against these compounds. Recombinant butyrylcholinesterase has been produced at a conc. of 5 g/L in the mammary gland of transgenic mice and goats (Huang et al., 2007). Transgenic goats can produce more than sufficient butyrylcholinesterase to protect all human beings at risk of organophosphorus poisoning. Some gene constructs have failed to produce economically significant amounts of protein in the milk of transgenic animals signifying that the technology still needs further refinement to assure consistent high-level expression. This is true for genes having complex regulation, such as those coding for erythropoietin (EPO) or human clotting factor VIII (hFVIII) (Hyttinen et al., 1994; Massoud et al., 1996; Niemann et al., 2000). The uses of somatic nuclear transfer will hasten the production of transgenic animals for mammary gland specific synthesis of recombinant proteins. 5. Increasing meat quantity Myostatin (MSTN) gene is a muscle-specific member of the transforming growth factor-beta (TGF-b) superfamily. The MSTN has a major role in the regulation of skeletal muscle growth. It is expressed predominantly in skeletal muscle and acts as a negative regulator of skeletal muscle growth by suppressing the proliferation and differentiation of myoblasts. Loss of function mutations in the MSTN gene is associated with increased skeletal muscle, commonly known as “double muscling.” This kind of loss of function mutations is found to occur naturally in some cattle breeds like Belgian Blue, Piedmontese, South Devon, and Asturiana de los Valles. Strategies have been developed to manipulate MSTN expression in order to improve the growth performance of livestock. dsRNA has been used to induce sequence-specific post-transcriptional gene silencing (PTGS) by using siRNA or shRNA. MSTN gene knockdown using RNAi has potential benefits for livestock production, as observed in the generation of transgenic sheep. This increases live weight, accelerate weight gain, increase carcass weight, and improve feeding efficiency. Additionally, MSTN gene knockdown approaches can also be utilized in chicken with the hope that it might help in producing transgenic chickens with enhanced muscle mass (Dushyanth et al., 2016). 6. Improving milk quality Suitable changes in ratios of milk proteins or the introduction of novel proteins into bovine milk can lead to an improvement in prevailing products. The RNAi approach has been explored to enhance the concentration of valuable components in milk (e.g., casein), as well as removing undesirable components (e.g., lactose) in cow’s milk. Apart from traditional dairy products, it has become possible to produce fat-free milk or milk with a modified lipid composition by means of modulation of enzymes involved in lipid metabolism; to increase curd or cheese production by increasing expression of the casein gene family in the mammary gland; to create “hypoallergenic” milk by knocking out b-lactoglobulin gene; to generate lactose-free milk via knockout of the a-lactalbumin locus; to produce “infant milk” in which more human lactoferrin is available. Cow’s milk contains heat-labile whey protein b-lactoglobulin, which is known to have allergenic properties. Many people suffer from milk allergy when they take cow milk. It is assumed that milk depleted of this b-lactoglobulin protein would be a better source for human consumption. This can be achieved by using anRNAi approach, by which, either, the disruption of b-lactoglobulin gene using Homologous Recombination (HR) or the specific knockdown of its expression can be done. The research demonstrated that b-lactoglobulin downregulation at the same time increases the k-casein and b-casein in milk significantly in a transgenic cow with targeted miRNA against b-lactoglobulin. Thus, the b-lactoglobulin depleted milk not only has potentially hypoallergenic properties, but its high casein content provides for increased calcium levels and high cheese yields also. The major portion of the adult population suffers from intestinal disorders due to lactose indigestion. It results from the physiological downregulation of the intestinal lactose-hydrolyzing enzyme at weaning. Hence, the supply of low lactose milk remains a possible solution. The knockdown of a-lactalbumin expression through RNAi offers a better control to achieve a satisfactory reduction of the lactose and water content of milk without affecting its vital attributes. Therefore RNAi offers an opportunity to reduce the lactose content and at the same time appreciably lower the transportation costs of milk. 7. Bioreactors of humanized organs With the advent of newer technologies in place, the therapeutic scope of RNA has stretched from the actual concept of RNA vaccine to its application in pharmaceutical and Tissue Engineering (Balmayor and Evans, 2019). The potential application of RNAi is regenerative medicine. In the field of biomedical application, malfunctioned organs are replaced with a new freshly harvested functional one. A large number of people are dying every year because of a lack of

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replacement organs, mostly in the case of the heart, liver, and kidney. Transgenic pigs have the potential to alleviate this short-fall since pig remains a preferred animal of choice as a bioreactor for harvesting xenogenic organs (Liu et al., 2016). But studies have shown that the use of pig derived human tissues is hampered by an unwanted porcine enzyme a(1,3) galactosyltransferase. This principal cell-surface xenoepitope is a primary target of the body’s natural antibody, and hence, is a key determinant in Hyper Acute Rejection (HAR). So, in order to avoid these glycosylation related problems, serious efforts are being made to create transgenic pigs that would enable tissue-specific silencing of a (1,3) galactosyltransferase. This would lead to the production of organs suitable for transplantation in humans and other primates. 8. Role of Transgenic animals in agriculture An important step toward the production of healthier pork has been made by the first pigs transgenic for a desaturase gene derived either from spinach or Caenorhabditis elegans. These pigs produced a higher ratio of polyunsaturated versus saturated fatty acids in their muscles, clearly indicating the potential of rendering more healthier pork in the near future (Niemann, 2004; Saeki et al., 2004; Lai et al., 2006). A human diet rich in nonsaturated fatty acids is correlated with a reduced risk of stroke and coronary diseases (Dewey et al., 2016). In the pig, it has been shown that transgenic expression of a bovine lactalbumin construct in sow milk resulted in higher lactose contents and greater milk yields, which correlated with better survival and development of the piglets (Donovan et al., 2001). The increased survival of piglets at weaning provides significant benefits to animal welfare and the producer. Phytase transgenic pigs have been developed to address the problem of manure-based environmental pollution. These pigs carry a bacterial phytase gene under the transcriptional control of a salivary gland specific promoter, which allows the pigs to digest plant phytate. Without the bacterial enzyme, the phytate phosphorus passes undigested into manure and pollutes the environment. With the bacterial enzyme, the fecal phosphorus output was reduced up to 75% (Golovan et al., 2001). These environmentally friendly pigs are expected to enter commercial production chains within the next few years. The physicochemical properties of milk are mainly affected by the ratio of casein variants, making them a prime target for the improvement of milk composition. The bovine casein ratio can be altered by overexpression of b- and k-casein, clearly underpinning the potential for improvements in the functional properties of bovine milk (Brophy et al., 2003). Mastitis is a preeminent health problem in modern dairy cattle production, causing significant economic losses. Lysostaphin has been shown to confer specific resistance against mastitis caused by Staphylococcus aureus. Cows have been cloned from transgenic donor cells that express a lysostaphin gene construct in the mammary gland.

7.12 RNAi in functional genomics A variety of methods has been used to knockdown genes via siRNA in mammalian species. RNAi is used in studies aimed at gene silencing in many species, including model species such as D. melanogaster and mammalian species. The RNAi gene libraries have also been generated in humans (Hu et al., 2009).

7.12.1 RNA therapeutics The United States Food and Drug Administration (FDA) approved two antisense oligonucleotide drugs in 2016, which can act on the mRNA of genes involved in incurable neurodegenerative diseases(Lieberman et al., 2018). And in 2017 itself, numerous synthetic RNAi drugs have undergone phase 2-3 of human clinical trials (Titze-de-Almeida et al., 2017). At present, a large number of clinical trials mainly focus on the use of miRNAs in disease diagnostics, as well as therapy (Kalariya et al., 2017). RNAi is a powerful approach for reducing the expression of endogenously expressed proteins. Live-attenuated vaccines are effective ways to establish robust, long-lasting immunity against viral infections. RNAi induced by short hairpin RNA (shRNA) has been found to be effective in controlling the Avian Influenza Virus (AIV) in a gene-specific manner (Betakova and Svancarova, 2017). Gene silencing mediated by siRNA serves as antiviral defense mechanisms in eukaryotes. siRNA duplexes targeted against Rift Valley fever virus (RVFV) nucleoprotein effectively inhibit the replication of RVFV in human and African green monkey cell lines. The individual or complex siRNAs, targeting the RVFV nucleoprotein gene, completely inhibited viral protein expression and prevented the degradation of host innate antiviral factor, the protein kinase R (PKR). In addition, pretreatment of cells with the nucleoprotein-specific siRNAs markedly reduced the virus load. It was inferred that the antiviral activity of RVFV nucleoprotein specific siRNAs might help in developing novel therapeutic strategy against infectious RVFV (Faburay and Richt, 2016). However, the possibility of reversion of live-attenuated vaccines, including miRNA targeted vaccines to wild-type replication and associated pathogenicity, are the possible threats associated with live-attenuated vaccines (Fay and Langlois, 2018). The shRNA targeting

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the noncoding region of the viral RNA of nucleoprotein might be a supporting tool in developing influenza-resistant chicks (Fujimoto et al., 2019). Recent successes in applying RNAi-based antiviral therapies in chickens highlight the usefulness of this approach in other species. Either in vitro or in vivo, bird cells have now been genetically engineered to resist viral diseases such as Marek’s disease, infectious bursal disease, avian leucosis, Rous sarcoma, and avian influenza (Hu et al., 2002; Li et al., 2005; Chen et al., 2007; Zhou et al., 2009; Lambeth et al., 2009; Wang et al., 2009).

7.12.2 Molecular insights into stem cell biology and genetic engineering Embryonic Stem Cells (ESCs) is one of the outstanding features as they are pluripotent and self-renewal under in vitro, as well as in vivo conditions. A detailed understanding of molecular mechanisms of biological properties of stem cells, and the mechanisms involved in the reprogramming of the cells and their differentiation into other cell lineages are of significant interest. RNAi technology has contributed immensely to revolutionize the functional genetics in animal cells. The genome-wide screening of RNAi has provided ample information about key scientific areas such as ESC biology, transcription factors, posttranslational modulators, and chromatin remodelers (Zheng and Hu, 2014). RNAi technology along with Sleeping Beauty transposon system constitutes a suitable method to produce transgenic cells that can be used to generate nuclear transfer cloned transgenic animals. By using these combinations, transgenic sheep, resistant to foot-andmouth disease, were generated (Deng et al., 2017).

7.12.3 Toward environmentally friendly farm animals Phosphorus pollution by animal production is a serious problem in agriculture, and excess phosphate from manure promotes eutrophication. Phytase transgenic pigs have been developed to address the problem of manure-based environmental pollution. Genetically engineered Enviropigs carry a bacterial phytase gene under the transcriptional control of a salivary gland specific promoter, which allows the pigs to digest plant phytate. Without the bacterial enzyme, the phytate phosphorus passes undigested into manure and pollutes the environment. With the bacterial enzyme, these environmentalfriendly pig has reduced the fecal phosphorus output up to 75% (Golovan et al., 2001).

7.13 Challenges RNAi is a promising technology in understanding functional genomics. It is rapidly becoming a method of choice for analyzing gene functions and holds promise for developing therapeutic agents based on the phenomenon of gene silencing (Balmayor and Evans, 2019). The progress in RNAi has shown promises for use in reverse genetic and therapy. RNAi is used to decipher the basic regulatory mechanism of gene regulation and modulation. One of the major challenges associated with RNAi is the mechanistic complexities that are only feebly understood until now. In addition, RNA is unstable and degraded by ribonucleases (RNases) that are prevalent in nature. It is difficult to deliver RNA across hydrophobic cell membranes. In general, miRNA targeting is a proficient platform for developing safe, effective vaccines and provides better plasticity over traditional live-attenuated vaccine strategies. As siRNAs do not integrate into the host genome, the RNAi response from siRNA is transient. RNAi-based targeted gene silencing is a quick, cost-effective, and reliable method to study gene expression, the function of the genes in cells or organisms. The effect of such specific but low knockdown can be masked by the off-target signature with phenotypic changes being undetectable. These limitations can make RNAi unpredictable, slow, and risky. RNAi technology combined with CRISPR/Cas9 provides the opportunity to produce genetically modified animals that are resistant to viral infection. This will minimize the economic losses to commercial livestock companies due to animal mortalities or the culling of infected animals (Xie et al., 2018). It is, therefore, necessary that transfection methods for applying RNAi should have minimal side effects.

7.14 Conclusions Small noncoding RNAs such as miRNAs involved in post-transcriptional regulation of gene expression orchestrate a wide range of functional biological and pathological processes. Efforts are made to ensure the gain of function through transgene integration into the livestock genome. Although the scope of gene silencing is relatively less, yet its potential is tremendous. Delivery of siRNA or shRNA construct remains a bottleneck for a wide application of this technology to generate transgenic animals. Therefore, attentions are shifted from transient siRNA delivery to stable siRNA (shRNA) cassette integration for constitutive gene silencing. The use of siRNA to knockdown gene expression in ESCs may provide a novel screening approach to understand gene function that is amenable for large-scale high throughput methods. RNAi

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causes disruption of gene expression in order to determine gene function or its effects on the metabolic pathway. The process has been used to study stem cells and prevent animal health by controlling parasites, pests, and pathogens. A multilevel control system adds more stringency to the regulatory aspect, and thus, helps to switch on and switch off a particular gene or set of genes in order to have a desired trait or product. Sometimes a gene may have an important function for a particular organ or tissue type, whereas, on other tissue it may not have any identified role. So inactivation or silencing of it may cause an adverse effect on health. Hence, tissue-specific gene silencing strategies become attractive. Although the therapeutic proteins derived from transgenic animals are well accepted by many people, using transgenic technology for improvements in animal productivity is not still accepted in many countries because of public concerns about Genetically Modified (GM) organisms. By considering the rapid advancement in gene targeting and transgenic technology, it is hoped that more the public would be aware of scientific reality about transgenic technology.

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Proc. Natl. Acad. Sci. Unit. States Am. 101 (17), 6361e6366. Schnieke, A.E., Kind, A.J., Ritchie, W.A., Mycock, K., Scott, A.R., Ritchie, M., Wilmut, I., Colman, A., Campbell, K.H.S., 1997. Human factor IX transgenic sheep produced by transfer of nuclei from transfected fetal fibroblasts. Science 278, 2130e2133. Shi, Y., Berg, J.M., 1995. A direct comparison of the properties of natural and designed zinc-finger proteins. Chem. Biol. 2, 83e89. Singh, A., Nie, H., Ghosn, B., Qin, H., Kwak, L.W., Roy, K., 2008. Efficient modulation of T-cell response by dual-mode, single-carrier delivery of cytokine-targeted siRNA and DNA vaccine to antigen-presenting cells. Mol. Ther. 16, 2011e2021. https://doi.org/10.1038/mt.2008.206. Singh, A., 2019. Introductory chapter: modulating gene expression-abridging the RNAi and CRISPR-cas9 technologies. In: Modulating Gene ExpressionAbridging the RNAi and CRISPR-Cas9 Technologies. IntechOpen. Singhal, M., Kansara, N., 2010. Transgenic animals: production and application. Int. J. Pharmaceut. Sci. Res. 1 (9), 12e22. Smithies, O., Gregg, R.G., Boggs, S.S., Koralewski, M.A., Kucherlapati, R.S., 1985. Insertion of DNA sequences into the human chromosomal betaglobin locus by homologous recombination. Nature 317, 230e234. Sokol, D.L., Murray, J.D., 1996. Antisense and ribozyme constructs in transgenic animals. Transgenic Res. 5, 363e371. Song, X., Evel-Kabler, K., Rollins, L., Aldrich, M., Gao, F., Huang, X.F., Chen, S., 2006. An alternative and effective HIV vaccination approach based on inhibition of antigen presentation attenuators in dendritic cells. PLoS Med. 3, e11. Stewart, C.R., Karpala, A.J., Lowther, S., Lowenthal, J.W., Bean, A.G., 2011. Immunostimulatory motifs enhance antiviral siRNAs targeting highly pathogenic avian influenza H5N1. PloS One 6, e21552. https://doi.org/10.1371/journal.pone.0021552. Stoppani, E., Bassi, I., Dotti, S., Lizier, M., Ferrari, M., Lucchini, F., 2015. Expression of a single siRNA against a conserved region of NP gene strongly inhibits in vitro replication of different Influenza A virus strains of avian and swine origin. Antivir. Res. 120, 16e22. https://doi.org/10.1016/ j.antiviral.2015.04.017. Tang, G., B. J., Reinhart, D.P., Bartel, P. D., Zamore, 2003. A biochemical framework for RNA silencing in plants. Genes Dev. 17, 49e63. Tenenhaus Dann, C., Alvarado, A.L., Hammer, R.E., Garbers, D.L., 2006. Heritable and stable gene knockdown in rats. Proc. Natl. Acad. Sci. U.S.A. 103, 11246e11251. Titze-de-Almeida, R., David, C., Titze-de-Almeida, S.S., 2017. The race of 10 synthetic RNAi-based drugs to the pharmaceutical market. Pharmaceut. Res. 34 (7), 1339e1363. Trends Biotechnol 22(6),286e294. Van den Hout, J.M.P., Reuser, A.J.J., De Klerk, J.B.C., Arts, W.F., Smeitink, J.A.M., Van der Ploeg, A.T., 2001. Enzyme therapy for Pompe disease with recombinant human a-glucosidase from rabbit milk. J. Inherit. Metab. Dis. 24 (2), 266e274. van Dyck, P.C., 2003. A history of child health equity legislation in the United States. Pediatrics 112 (Suppl. 3), 727e730. Vaucheret, H., Fagard, M., 2001. Transcriptional gene silencing in plants: targets, inducers and regulators. Trends Genet. 17, 29e35. Wang, Y., Sun, H., Shen, P., Zhang, X., Xia, X., Xia, B., 2010. Effective inhibition of replication of infectious bursal disease virus by miRNAs delivered by vectors and targeting the VP2 gene. J. Virol Methods 165, 127e132.

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Wang, Y., Sun, H., Shen, P., Zhang, X., Xia, X., 2009. Effective inhibition of infectious bursal disease virus replication by recombinant avian adenoassociated virus-delivered microRNAs. J. Gen. Virol. 90 (6), 1417e1422. Whitelaw, C.B.A., Radcliffe, P.A., Ritchie, W.A., Carlisle, A., Ellard, F.M., Pena, R.N., Rowe, J., Clark, A.J., King, T.J., Mitrophanous, K.A., 2004. Efficient generation of transgenic pigs using equine infectious anaemia virus (EIAV) derived vector. FEBS Lett. 571, 233e236. Whyte, J.J., Zhao, J., Wells, K.D., Samuel, M.S., Whitworth, K.M., Walters, E.M., Laughlin, M.,H., Prather, R.S., 2011. Gene targeting with zinc finger nucleases to produce cloned eGFP knockout pigs. Mol. Reprod. Dev. 78 (2). Witter, R.L., 1997. Increased virulence of Marek’s disease virus field isolates. Avian Dis. 41, 149e163. Wongsrikeao, P., Sutou, S., Kunishi, M., Dong, Y.J., Bai, X., Otoi, T., 2011. Combination of the somatic cell nuclear transfer method and RNAi technology for the production of a prion gene-knockdown calf using plasmid vectors harboring the U6 or tRNA promoter. Prion 5, 39e46. Xie, Z., Pang, D., Yuan, H., Jiao, H., Lu, C., Wang, K., Yang, Q., Li, M., Chen, X., Yu, T., Chen, X., Dai, Z., Peng, Y., Tang, X., Li, Z., Wang, T., Guo, H., Li, L., Tu, C., Lai, L., Ouyang, H., 2018. Genetically modified pigs are protected from classical swine fever virus. PLoS Pathog. 14 (12), e1007193. Xie, Z.,E., Allen, A., Wilken, J.C., C., 2005. DICER-LIKE 4 functions in trans-acting small interfering RNA biogenesis and vegetative phase change in Arabidopsis thaliana. Proc. Natl. Acad. Sci. U.S.A. 102, 12984e12989. Xu, Y.-F., Shen, H.-Y., Zhao, M.-Q., Chen, L.-J., Li, Y.-G., Liao, M., Jia, J.-T., Lv, Y.-R., Yi, L., Chen, J.-D., 2012. Adenovirus-vectored shRNAs targeted to the highly conserved regions of VP1 and 2B in tandem inhibits replication of foot-and-mouth disease virus both in vitro and in vivo. J. Virol Methods 181, 51e58. Zheng, X., Hu, G., 2014. Use of genomewide RNAi screens to identify regulators of embryonic stem cell pluripotency and self-renewal. Methods Mol. Biol. 1150, 163e173. Zhou, Y.J., Yu, H., Tian, Z.,J., Li, G.,X., Hao, X.,F., Yan, L.,P., Wei, T.,C., 2009. Genetic diversity of the ORF5 gene of porcine reproductive and respiratory syndrome virus isolates in China from 2006 to 2008. Virus Res. 144 (1e2), 136e144. Ziomek, C.,A., 1998. Commercialization of proteins produced in the mammary gland. Theriogenology 49 (1), 139e144.

Further reading Carneiro, I.S., Menezes, J.N.R., Maia, J.A., Miranda, A.M., Oliveira, V.B.S., Murray, J.D., Maga, E.A., Bertolini, M., Bertolini, L.R., 2018. Milk from transgenic goat expressing human lysozyme for recovery and treatment of gastrointestinal pathogens. Eur. J. Pharmaceut. Sci. 15 (112), 79e86. Crispo, M., Mulet, A.P., Tesson, L., Barrera, N., Cuadro, F., 2015. Efficient generation of myostatin knockout sheep using CRISPR/Cas9 technology and microinjection into zygotes. PloS One 10, e0136690. DiCarlo, J.E., Norville, J.E., Mali, P., Rios, X., Aach, J., Church, G.M., 2013. Genome engineering in Saccharomyces cerevisiae using CRISPR-Cas systems. Nucleic Acids Res. 41, 4336e4343. Fire, A., 1999. RNA triggered gene silencing. Trends Genet. 15, 358e363. Hwang, W.Y., Fu, Y., Reyon, D., Maeder, M.L., Kaini, P., et al., 2013. Heritable and precise zebrafish genome editing using a CRISPR-Cas system. PloS One 8, e68708. Jiang, W.1., Bikard, D., Cox, D., Zhang, F., Marraffini, L.A., 2013. RNA-guided editing of bacterial genomes using CRISPR-Cas systems. Nat. Biotechnol. 31 (3), 233e239. Karatzas, C.N., 2003. Designer milk from transgenic clones. Nat. Biotechnol. 21, 138e139. Lv, Q., Yuan, L., Deng, J., Chen, M., Wang, Y., Zeng, J., Li, Z., Lai, L., 2016. Efficient generation of myostatin gene mutated rabbit by CRISPR/Cas9. Sci. Rep. 6, 25029. Ma, T., Tao, J., Yang, M., He, C., Tian, X., Zhang, X., Liu, G., 2017. An AANAT/ASMT transgenic animal model constructed with CRISPR/Cas9 system serving Bosze as the mammary gland bioreactor to produce melatonin-enriched milk in sheep. J. Pineal Res. 63 (1), e12406. Mali, P., Esvelt, K.M., Church, G.M., 2013. Cas9 as a versatile tool for engineering biology. Nat. Methods 10, 957e963. Nims, S.D., Porter, C.A., Midura, P., Palacios, M.J., Ayres, S.L., Denniston, R.S., Hayes, M.L., Ziomek, C.A., Meade, H.M., Godke, R.A., Gavin, W.G., Overstrom, E.W., Echelard, Y., 1999. Production of goats by somatic cell nuclear transfer. Nat. Biotechnol. 17 (5), 456e461. Parc, A.L., Karav, S., Rouquie, C., Maga, E.A., Bunyatratchata, A., Barile, D., 2017. Characterisation of Recombinant Human lactoferrin N-N- glycans expressed in the milk of transgenic cows. PloS One 12 (2), e0171477. Proudfoot, C., Carlson, D.F., Huddart, R., Long, C.R., Pryor, J.H., King, T.J., Lillico, S.G., Mileham, A.J., McLaren, D.G., Whitelaw, C.B., Fahrenkrug, S.C., 2015. Genome edited sheep and cattle. Transgenic Res. 24 (1), 147e153. Reh, W.A., Maga, E.A., Collette, N.M., Moyer, A., Conrad-Brink, J.S., Taylor, S.J., Murray, J.D., 2004. Hot topic: using a stearoyl-CoA desaturase transgene to alter milk fatty acid composition. J. Dairy Sci. 87, 3510e3514. Richt, J.A., Kasinathan, P., Hamir, A.N., Castilla, J., Sathiyaseelan, T., Vargas, F., Sathiyaseelan, J., Wu, H., Matsushita, H., Koster, J., Kato, S., Ishida, I., Soto, C., Robl, J.M., Kuroiwa, Y., 2007. Production of cattle lacking prion protein. Nat. Biotechnol. 25, 132e138. Singh, V., Braddick, D., Dhar, P.K., 2017. Exploring the potential of genome editing CRISPR-Cas9 technology. Gene 599, 1e18. Tessanne, K., Golding, M.C., Long, C.R., Peoples, M.D., Hannon, G., Westhusin, M.E., 2012. Production of transgenic calves expressing an shRNA targeting myostatin. Mol. Reprod. Dev. 79 (3), 176e185. Tuschl, T., Borkhardt, A., 2002. Small interfering RNAs, a revolutionary tool for the analysis of gene function and gene therapy. Mol. Interv. 2, 158. Van Berkel, P.H., Welling, M.M., Geerts, M., van Veen, H.A., Ravensbergen, B., Salaheddine, M., Pauwels, E.K., Pieper, F., Nuijens, J.H., Nibbering, P.H., 2002. Large scale production of recombinant human lactoferrin in the milk of transgenic cows. Nat. Biotechnol. 20, 484e487.

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Wang, H., Yang, H., Shivalila, C.S., Dawlaty, M.M., Cheng, A.W., Zhang, F., Jaenisch, R., 2013. 0ne-step generation of mice carrying mutations in multiple genes by CRISPR/Cas-mediated genome engineering. Cell. 153 (4), 910e918, 2013 May 9. Wang, T., Wei, J.J., Sabatini, D.M., Lander, E.S., 2014. Genetic screens in human cells using the CRISPR-Cas9 system. Science 343, 80e84. William, A., Ritchie, T.K., Neil, C., Ailsa, J., Carlisle, S.L., Gerry McLachlan, C., Bruce, A., Whitelaw, 2009. Trans. sheep design. transplant. studies 76 (Issue1), 61e64. Wu, H., Wang, Y., Zhang, Y., Yang, M., Lv, J., Liu, J., 2015. TALE nickase-mediated SP110 knockin endows cattle with increased resistance to tuberculosis. Proc. Natl. Acad. Sci. Unit. States Am. 112 (13), E1530eE1539. Yu, C., Liu, Y., Ma, T., Liu, K., Xu, S., Zhang, Y., Liu, H., La Russa, M., Xie, M., Ding, S., Qi, L.S., February 5, 2015. Small molecules enhance CRISPR genome editing in pluripotent stem cells. Cell Stem Cell 16 (2), 142e147.

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Chapter 8

Microbial metagenomics: potential and challenges Shikha1, Shailja Singh1 and Shiv Shankar2 1

Department of Environmental Science, Babasaheb Bhimrao Ambedkar University, Lucknow, Uttar Pradesh, India; 2Department of Environmental

Science, School of Vocational Studies and Applied Sciences, Gautam Buddha University, Greater Noida, Uttar Pradesh, India

8.1 Introduction The metagenomic analysis is generally employed to investigate complex microbial communities directly sampled from the environment without formal isolation and culture of any organism. Microbes are known to play important roles in different ecosystems; however, most of them remain uncharacterized in detail (Riesenfeld et al., 2004). “Metagenomics” coined in 1998, has been found helpful in understanding multifarious aspects of an environmental sample, allowing one to characterize the microorganisms in the given sample (Evangelou and Ioannidis, 2013). It is not only helpful in the identification and characterization of diverse species occurring in a community but also provides an insight into the metabolic and functional aspects of the microbes in the environmental sample. Contemporary metagenomic approaches elucidate the complexities of the microbiota and their functional dynamics in the natural ecosystem. Different metagenomic approaches are addressed to resolve fundamental questions related to taxonomic diversity, and the roles played by them (Functional metagenomics) (Almeida et al., 2019). PCR (Polymerase chain reaction) amplification of some specific target genes like 16S rRNA, 18S rRNA, Nif H, ribosomal ITS etc., prior to sequencing allows diversity analysis and assessment of relative abundance of a particular microorganism in a community. The most abundant microorganism in a specific group may not necessarily be associated with the specificity of that group with reference to the functioning of the community. Similarly, the most abundant organisms in a community may not play the most critical role in the community; however, organisms constituting merely 0.1% of the community may reveal important functions such as nitrogen-fixation (Rocha-Martin et al., 2014). Metagenomic techniques require extraction of DNA from a community so that genomes of all the organisms in that community can be pooled. Metagenomics thus involves genomic analysis of microbes by direct extraction and cloning of DNA from an assemblage of microorganisms inhabiting any existing environments on earth like water or the soil. Usually, the genomes are isolated from an environment and fragmented, followed by cloning into an organism of choice through their plasmid that has the capacity to replicate (Zaouri et al., 2019). Such organisms are subsequently cultured to form metagenomic libraries that can be subjected to DNA-based sequencing for further analysis. Direct isolation of nucleic acids from environmental samples has been found to be a powerful tool for ecological comparisons and exploration (Handelsman, 2004). The idea of cloning DNA directly from the environmental samples was given by Pace and his coworkers in 1985. In 1991, Schmidt and his colleagues used this approach for cloning DNA from picoplankton in a phage vector and subjected it for subsequent 16S rRNA gene sequence analyses. The first successful function-driven metagenomic libraries were screened in the year 1995 and termed as zoo libraries. However, the term was coined in 1998 by Jo Handelsman and his colleague, defined as “the genomic analyses of microorganism by direct extraction and cloning of DNA from an assemblage of microorganism.” This technique rendered access to the majority of organisms that were unculturable by conventional methods (Schmidt, 1991).

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8.2 Metagenome and metagenomics Metagenome represents the community genome and not the microbial community present in a given environmental sample. Metagenomics includes strategic investigations at three different levels that are mutually interconnected viz., processing the sample, and sequencing of DNA followed by functional analysis, which aims to get a global view of the function of the microbial world (Garza, 2015).

8.2.1 Habitat selection The choice of a microbial community to study depends upon the scientific question lying underneath. Significant information about the habitat, be it physical, chemical, or ecological, gives more insight into the metagenomic data. Discovery of a keystone species (a member of a community whose significance to the community is larger than its relative abundance) relies on knowledge of the site (Alves, 2018).

8.2.2 Sampling The samples (Type, size, scale, number) must be representative of the habitats. Community’s response to changing conditions with time, community structure that is critical to understand, function, and robustness, all play a significant role in metagenomic recovery.

8.2.3 Macromolecule recovery The quality and completeness of data obtained from metagenomic analysis of any community will be only as good as the procedures used for the extraction of DNA from a sample. Cells from different species differ in their susceptibility to lysis under various conditions; even members of the same species may differ in their susceptibility to lysis in different physiological states; DNA from some members may be degraded DNA from dead cells (Nelson et al., 2019).

8.3 Next-generation sequencing (NGS) to explore microbial communities Earlier Sanger sequencing technology was used to study microbial communities. But, nowadays, both, the sequencing yield and the sequence length, have significantly changed. Presently, approximately 96 sequences per run, having an average length of 650 bp, can be retrieved through Sanger sequencing, which may be considered adequate for analysis of phylogenetic markers. In the above context, Next-Generation Sequencing (NGS) technologies have emerged as low-cost platforms having the capability of sequencing millions of DNA molecules in parallel, having different yields, as well as sequence lengths elaborating a favorable impact in diversified areas (Fichot and Norman, 2013; Sanchez-Flores and AbreuGoodger, 2014).

8.3.1 Pyrosequencing The NGS technology that revolutionized both the genomics, as well as metagenomics, was “pyrosequencing,” alternatively depicted as the 454 sequencing platform. This technology works on the principle of addition of a one-by-one nucleotide in a cycle, wherein the pyrophosphate (PPi) liberated from the reaction of DNA polymerization gets transformed into a luminous signal, and the light emission from a given DNA fragment is detected and translated to nucleotide sequences with the help of machine with further gives corresponding base quality value. Pyrosequencing offers a greater yield with shorter read lengths compared to Sanger sequencing and that too, at a lower cost. Although this technology is now obsolete owing to artificial insertions and deletions because of long homopolymeric regions; however, the entire software that was developed to analyze 454 data might be adapted to analyze data that has been obtained from other platforms (Margulies et al., 2005).

8.3.1.1 Ion Torrent sequencing The Ion Torrent sequencing emerged as an analogous technology to the 454 sequencing platform that produces almost similar yield and a read length. The technology consists of the smallest existing potentiometer that can detect any change in hydrogen potential that is generated each time a proton is released subsequent to the addition of a nucleotide in the sequencing reaction taking place in millions of microwells. The maximum yield is recorded as w500 million reads having

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a mode length of 400 bp. The technology is cost-effective since the Ion Torrent platform is just one 10th of the pyrosequencing cost (Rothberg et al., 2011; Glenn, 2014).

8.3.1.2 Illumina technology The Illumina technology, over the period, has become one of the most appealing technologies owing to its low cost and greater yield. The principle behind Illumina chemistry is reversible-termination sequencing through the synthesis of fluorescently labeled nucleotides. In a flow cell, the DNA fragments are attached and distributed, wherein the sequencing reaction takes place by the addition of a labeled nucleotide. Subsequent to the incorporation of the labeled nucleotide, an excitation of its fluorescent molecule is incited by a laser beam, and the signal gets registered by the machine. Later on, the next nucleotide may be incorporated after the removal of the fluorophore molecule. Sequencing of the DNA fragments can be achieved either from one or both sides resulting in a single end or pair-end sequence, respectively, with a read length of maximum 300 base pairs per read. Presently among the second-generation sequencing technologies, the output achieved out of this technology is the highest, rendering it suitable for mixing hundreds of samples (Bennett, 2004).

8.3.1.3 PacBio RS and Oxford Nanopore The above-mentioned technologies are widely used for metagenome projects; however, the evolution of sequencing continues to resolve the known constraints of these technologies in terms of better yield, cost-effectiveness, and read length. Currently, the third-generation sequencing technologies include PacBio RS from Pacific Bioscience and the Oxford Nanopore (Kasianowicz et al., 1996). These are single-molecule, real-time technologies that have reduced the amplification constraint, as well as the short read length problem. These technologies have reduced both time and cost, which is much valued. Although, as compared to other technologies, the error rate is higher but can be corrected by increasing the sequencing depth. Moreover, if considered in terms of computational tools, virtually no software exists that may be used for metagenomics-based analysis (Fichot and Norman, 2013). Another great evolution of both second- and third-generation sequencing technologies is that it does not require either DNA cloning vectors or bacterial hosts for library preparation, thereby not only elucidating the library preparation but also reducing DNA contamination from other sources that do not form the part of the metagenome. The new generation sequencing technologies apart from their contribution toward the discovery of novel microbial worlds and biomolecules have given the ease to explore new environments. However, these technologies reflect specific limitations and constraints that need to be circumvented. Data obtained from second- or third-generation sequencing technologies require computational analysis. The larger the dataset, the higher are the computational resources necessitating complex bioinformatics. Bioinformatic analysis requires high-end servers along with UNIX operative system skills. In order to run and install the metagenomics software programming, as well as scripting, knowledge is desired to ease parsing and interpretation of the results. Hence, biologists or biological scientists need to develop basic computational skills to get the maximum output of metagenomic data (Logares et al., 2012).

8.3.2 Reconstructing the genomic content of the microbial community from NGS data Microbial diversity can be ascertained following two different approaches: (1) Amplicon sequencing or (2) Shotgun metagenomics. In Amplicon sequencing, amplification of specific regions of DNA obtained from communities is carried out using taxonomically informative primer targets like the 16S rRNA gene (in prokaryotes) and intergenic transcribed spacers (ITS) or the large ribosomal subunit (LSU) gene (in eukaryotes). On the other hand in, shotgun metagenomics, large fragments or complete genomes are reconstructed from DNA in a community without prior isolation, which allows characterization of a large number of both coding and noncoding sequences that might be used as phylogenetic markers (Tonge et al., 2014).

8.3.3 Amplicon sequencing analyses The term “metagenomics” could not be employed to state amplicon sequence analysis since this analysis depends upon a single gene rather than a collection of all the genes available in the genomes of all the organisms in a sample. A better term for amplicon sequence analysis is “metaprofiling,” and refers to study all the members in a specific microbial community depending upon one gene or marker (i.e., 16S rRNA gene) for phylogenetic purposes. Metaprofiling can be conveniently used for taxonomic and phylogenetic classification in such samples that are large and complex and within organisms underlying different life domains employing almost all of the abovementioned sequencing

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technologies. Metaprofiling is one of the best options for 16S rRNA amplicon library preparation and sequencing employing Illumina, MiSeq, or Ion Torrent (Luo et al., 2012). The major disadvantage of amplicon sequencing arises from the constraint generated from using a single phylogenetic marker (like 16S ribosomal gene) or another variable region. Other drawbacks include low resolution specially at the species level (Nalbantoglu et al., 2014), horizontal transfer of 16S rRNA genes (Bodilis et al., 2012), besides the fact that merely < 0.1% of the total genome happens to be the ribosomal genes, which hinder the amplification of this marker from scanty genomes in a sample. For more than the last 4 decades the ribosomal genes have been used as phylogenetic markers; as a result, this marker has achieved wide representation in many databases, due to which the taxonomic annotation of the microorganisms present in any metagenomic sample gets possible. Greengenes, the Ribosomal Database Project, and the Silva are some of the examples of such databases (DeSantis et al., 2006; Wang et al., 2007; Quast et al., 2013). The amplicon-dependent techniques are very much prone to sequencing errors since result discrepancy has been obtained when different ribosomal variable regions or primers are used, besides OTU assignment errors (Poretsky et al., 2014).

8.3.4 Shotgun metagenomics Subsequent to the deciphering of the microbial diversity of a metagenome, a greater understanding of its metabolic potential is achieved. This can be accomplished through the whole metagenome approach wherein total DNA is pooled to prepare shotgun libraries. The impact of shotgun metagenomics aids in taxonomic species level classification.

8.3.4.1 Assessment of taxonomy based on markers Through whole metagenome shotgun sequencing, representation of all the genomes in a given sample is obtained. This allows a choice among a wide range of phylogenetic markers so as to perform taxonomic annotation that may include the ribosomal or any other markers coming under the amplicon sequencing approach. Parallel-meta is a multithreading software to extract ribosomal marker genes from metagenomic sequences in order to carry out taxonomic annotation (Su et al., 2014). The program allows a Hidden Markov Models (HMM)-based reconstruction algorithm to collect the ribosomal sequences from short reads (De Fonzo et al., 2007). Using Megablast, the reconstructed sequences can be further mapped to different 16S gene databases. As discussed elsewhere, by using more than one phylogenetic marker, the taxonomical annotation could be further improved (http://www.ncbi.nlm.nih.gov/blast/ html/megablast.html). Therefore, through software, single-copy marker genes could be searched in other databases. MOCAT (Kultima et al., 2012) and AMPHORA (Wu and Eisen, 2008) are the two examples of programs using these approaches. MOCAT includes the RefMG database (Ciccarelli et al., 2006) made by a collection of 40 single-copy marker genes, while AMPHORA includes a database containing approximately 31 single-copy universal markers. Postsingle-copy marker identification, an OTU multiple sequence alignment is performed followed by, distance calculation, and clustering. The taxonomical annotation is finally performed using reference genomes that give species resolution.

8.3.4.2 The binning strategy Binning is a fast and practical method to determine taxonomical composition employing the information present in the reads. It can be carried out using either reads or assembled sequences. Different strategies are used by binning algorithms to get the taxonomic assignment: (i) sequence composition classification (ii) sequence alignment against appropriate references. The earlier one is dependent upon k-mer frequencies, which employs short words like k-mers to demonstrate a vectorlike sequence and subsequently to derive the similarity amongst all words within a query. Such kind of representation may be regarded as a “genomic signature” (Karlin and Burge, 1995) to establish evolutionary conservation within species. The examples of software employed to carry out sequence classification through composition include TETRA, PhylophytiaS, and MetaclusterTA (Teeling et al., 2012; McHardy et al., 2006; Wang et al., 2014). There are several other methods that employ more than one strategy to establish correct binning of sequences, for example, MaxBin and Amphora2 (Wu et al., 2014; Wu and Scott, 2012), which depends upon k-mer signatures, singlecopy marker genes, GC content, etc., to perform contig and read binning. Although the binning strategy facilitates taxonomic classification, it has some problems with horizontally transferred sequences, wherein genes from one organism appear in another organism, thereby leading to misclassification, especially between nondescribed organisms (Sharpton, 2014). However, depending upon the availability of long reads, it gets possible to undergo a taxonomic assignment by translating them and employing all potential coding sequences to carry out

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searches in annotated protein databases using local alignment tools (like BLAST). In the end, it could be considered that the availability of more information supporting taxonomic or functional results, conclusions become more reliable. Hence more than one approach should be used to assess taxonomic or functional annotation.

8.4 Bioprospecting of metagenomes Recovery of novel biomolecules from any environmental samples involves two techniques; a sequence-based (computational) technique and a function-based technique (experimental) (Fig. 8.1). Abovementioned both screening techniques comprise cloning of DNA from environmental samples and subsequent construction of small-insert/large-insert libraries. The resulting metagenomic libraries are used to transform a host (usually Escherichia coli) (Knietsch et al., 2003). Since significant differences in modes of expression exist between different taxonomic groups of prokaryotes and approximately 40% of the enzymatic activities has been claimed to be detected following random cloning in E. coli, additional hosts, such as Sulfolobus solfataricus, Streptomyces spp, Thermus thermophilus, and Proteobacteria have been used to widen the range of detectable activities under metagenomic screens (Simon and Daniel, 2010).

8.4.1 Sequence-based analyses The sequence-based analysis involves sequencing and analysis of DNA directly obtained from environmental samples. Also known as metagenome sequencing, this analysis is used to determine the DNA sequences of an entire metagenome. This type of analysis involves the designing of probes or primers of DNA derived from the conserved regions of preknown genes or protein families, thereby only novel variants of proteins having known functional classes can be identified. Sequence-based metagenomic studies can be employed to identify genes, assemble genomes, work out complete metabolic pathways, as well as to compare organisms belonging to different communities (Ramya et al., 2019). Genome assembly requires computational skills and power since it can offer a better understanding of how certain genes help organisms to survive in a particular (extreme) environment, e.g., one can find a specific species screened from an iron mine having genes involved in iron metabolism. Sequence-based metagenomics helps in establishing the extent of diversity and the magnitude of different bacterial species present in a particular sample. Analysis of microbial diversity is cost-effective and comparatively less computer-intensive compared to assembling genomes and may elaborate valuable information as regards to ecology of microbes in a given sample (Ghosh et al., 2019).

FIGURE 8.1 Sequence based and function based metagenomics.

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8.4.2 Function-based analyses This approach toward metagenomic analysis refers to the identification of clones that express a particular function. The success of this approach depends upon faithful transcription followed by the translation of the gene/genes of interest and secretion of the gene product, in case the screening or assay procedures mark it to be extracellular. Function-based analyses include the following steps: (i) DNA extraction, (ii) DNA cloning into a vector (a DNA molecule that carries foreign DNA into a suitable host cell). (iii) Transformation of clone into a suitable bacterium (Host cell) and finally (iv) Screening of transformants (Ngara and Zhang, 2018). This approach is unique in itself in that it does not require the genes of interest to be recognizable following sequence analysis since the identification of the entirely new classes of genes is through new or known functions. The significant limitation of this approach is that most of the genes may not get expressed in any specific host bacterium that has been selected for cloning (Østergaard and Yanofsky, 2004).

8.4.2.1 Metagenomic DNA extraction Isolation or extraction of DNA from environmental samples is a primary step for the above-mentioned metagenomic approaches. DNA extraction can be carried out following two methods: (i) direct DNA extraction that consists of cell lysis and (ii) indirect DNA extraction (Narayan et al., 2016). 8.4.2.1.1 Direct DNA extraction Direct DNA extraction initiates with the purification of DNA since many contaminants such as humic substances, etc., present in environment sample, need to be separated from DNA. DNA can also be purified on the basis of charge using electrophoresis techniques. Since both DNA, as well as the humic substances, happen to be negatively charged molecules, they tend to run along the same direction (i.e., toward cathode); however, positively charged molecules (contaminants) move in an opposite direction. On the other hand, DNA can also be purified on the basis of molecular weight following chromatography techniques like: ion exchange chromatography, size exclusion chromatography, etc. (Tan and Yiap, 2009). Extraction of DNA is done by adding glass beads to the sample in extraction buffer [Tris HCL (pH-8.0), 100 mM sodium EDTA (pH 8.0) and 1.5 M NaCl]. The sample is thoroughly blended for 2 min to which sodium dodecyl sulfate is added and subjected to incubation at 65
C for 1 h. Postincubation, the sample is centrifuged, and the lysate is then transferred to clean centrifuge tubes to which isopropanol is added and further kept at 4
C for 2 h. The sample is centrifuged once again, pellet washed with ethanol (70%), air dried and stored in Tris buffer. The presence of DNA in the sample is further confirmed using electrophoresis, and DNA is subjected to purification either on the basis of charge or following molecular methods. Physical disruption methods can produce a considerable yield of DNA but may cause shearing, which is not suitable for the construction of a large-insert metagenomic library. It is, therefore, suggested that enzymatic or chemical lyses can be employed instead of sonication or bead beating (Natarajan et al., 2016). 8.4.2.1.2 Freeze/thaw Cells can also be ruptured following freeze/thaw technique, wherein various combinations with buffers and enzymes are employed. To the sample, Tris-HCl buffer (Tris-HCl-100 mm, EDTA-100 mM and CTAB (cetyl trimethyl ammonium bromide) 0.1% at pH 8.0) is added. The sample is then repeatedly frozen and thawed thrice at þ65
C and 65
C for 30 min. The formation of ice crystals during freezing is well known to cause the rupture of the cells. 8.4.2.1.3 Indirect DNA extraction Sample are homogenized in Tris-HCl (100 mM), SDS (100 mM) and CTAB (0.1%), at pH 8.0, and further centrifuged to remove impurities. Through repeated blending, an adequate recovery of bacterial biomass can be achieved for DNA extraction. Supernatant collected during repeated cycles of blending is pooled, and the bacterial cells thus collected are concentrated by centrifugation (10,000  g; 30 min). Bacterial recovery can be checked using a microscope. Cells are further exposed to proteinase to inactivate proteins and enzymes postcells lysis. The indirect method happens to be timeconsuming but has less contamination. Although the process yields less amount of DNA, probably due to a specific target (prokaryotic DNA), a large DNA size is obtained. Reports are there in which bacterial cells have been separated using cation exchange resin. While comparing the above method, it could be derived that although the direct method (beading) results in large quantities of nucleic acids, the resulting nucleic acid extracts are generally sheared and contaminated with

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humic acids and often contain unknown amounts of extracellular and eukaryotic DNA. On the other hand, the indirect extraction method may be time consuming but revealed the highest DNA purity and low amount of DNA shearing. Hence, this method is more popular when targeting prokaryotic communities (Zitzmann et al., 2017).

8.4.2.2 Library preparation After isolation and purification of genetic material, a metagenomic library is constructed, which consists of cloning of the DNA fragments in specific vectors, which is then inserted into a suitable host cell. E. coli happens to be the universal host strain for cloning and expression of genes since its genome is well defined and could be easily transformed (Steele et al., 2009). Subsequently, screening for the genes and/or functions of interest is carried out. The identification of the functional clone from the metagenomic library is a challenging approach in itself. Transcription and/or translation of the genes of interest and the extracellular extraction assay of clones are very crucial and should be appropriately conducted (Gagic et al., 2016). Library preparation of environmental samples for shotgun metagenomic sequencing requires amplification. Certain samples (water, swabs) may yield meager amounts of DNA, thereby necessitating amplification during the library preparation. Amplification by PCR may result in an overamplification of certain fragments compared to other obfuscating measurements related to abundance and microbial diversity. Most of the time, one may not have any choice when low inputs of DNA is obtained. It is a good practice to minimize variability, constructing libraries together to reduce individual batch effects and keeping the steps of the library preparation as consistent as possible between samples as a good practice. If the extraction of enough DNA material (w250e500 ng) could be accomplished, an amplification-free method for the library is recommended (Thomas et al., 2012) (Fig. 8.2).

8.4.2.3 Screening Subsequent to the transformation of the clones into a host bacterium, the next important step is screening the resulting transformants. Screening involves either screening for expression of specific traits (e.g., enzymatic activity, antibiotic production) or identification of conserved gene following hybridization PCR using phylogenetic markers, e.g., 16 S rRNA (Cabral et al., 2019). Although the metabolic and functional capacity of the microbial community is displayed by metagenomics, it is also true that metagenomic-based DNA analysis does not have the ability to differentiate between expressed and unexpressed genes. Metagenomics may not give evidence toward the metabolic activity of the expressed genes. To this end, conventional reverse transcriptase quantitative PCR (RT-PCR) happened to be the main tool in order to detect and quantify various transcripts in the environment. However, in order to run the RT-PCR adequate primary knowledge of the targeted gene sequence is very important to design the primers. Environmental microarrays have been developed (highdensity array technology) to overcome the gene number constraints and/or analysis of mRNA derived cDNA clones libraries. Aforesaid approaches have given significant insights into the gene expression in a microbial community. However, the sensitivities in detection are unequal for the imprinted sequences, since results solely depend on the selected conditions for hybridization. Low-abundance transcripts could not be detected as often. Through transcript cloning, such types of problems could be avoided following random amplification and further mRNA fragments sequestration, but this initiates other problems related to the cloning system along with the host of the libraries. Direct cDNA sequencing using nextgeneration sequencing technologies can circumvent the limitations of both approaches addressed above, which may provide affordable access to the metatranscriptome, further allowing expression profiling of the whole-genome of a given microbial community (Parages et al., 2016).

8.5 Applications of metagenomics According to an estimate, less than 1% of the microorganisms existing in the natural environment could be cultured in the laboratory. A large number of natural products have been reported to exist in the unculturable microbes. The metabolic activities of the unculturable microbes find extensive applications in different industrial and biomedical applications (Sousa et al., 2019). Metagenomics gives an extensive resource for the evolution of novel genes, bioprocesses, natural products, enzymes, bioactive compounds, etc. that may considerably affect various industrial and biotechnological applications:

8.5.1 Biocatalysts and metagenomics Metagenomics has been found to be a well-proven and powerful approach to suffice the growing demand for novel biocatalysts and enzymes. Xylanases, proteases, lipases, amylases, cellulases, and several other important industrial

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FIGURE 8.2 Major steps for metagenomic library construction.

enzymes have known to be produced through metagenomics. Described below are some major enzymes that have been tapped from genetically unexploited resources (Distaso et al., 2017).

8.5.1.1 Xylanases Xylanases (endo- 1, 4-xylanases, EC 3.2.1.8) are key enzymes for xylan degradation. They cleave xylan backbone into smaller oligosaccharides, thereby breaking hemicellulose, a major component of the plant cell wall. These enzymes have a wide range of applications in a variety of industrial processes such as clarification of juices, extraction and preparation of beverages, generation of protoplast in plant cells, detergents, production of surfactants, production of polysaccharides that are pharmacologically active and find application as antimicrobial agents, antioxidants, etc. It has been reported that xylanases are produced by different microorganisms on different sources, which brings them greater importance with respect to the application. Xylanases have been detected in the metagenome variety of diverse environments, e.g., in an insect gut, an anaerobic digester (thermophilic), and dairy farm waste lagoon. Xylanases have been used in the conversion of biomass to biofuel and for the production of fermentable sugars. It has been found that xylanases isolated from the metagenomic library exhibited a clear zone of xylan hydrolysis on RBB plate containing xylan and displayed activity over a broad range of pH and temperature having optima at 9.0 and 80
C respectively, besides being highly thermostable expanding their industrial use. GH11 xylanase gene xyl7 has been reported to be derived from metagenomics. The metagenomically derived enzyme revealed high specific activity, greater stability, and more soluble protein yield proving it

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as an excellent candidate for variety of industrial application. The xylanase gene, xyn10J, isolated from the compost metagenomic library, was found to hydrolyze xylotetraose and xylohexaose to xylobiose and xylopentaose, respectively, while xylotriose was hydrolyzed to xylobiose having transglycosylation activity. The enzyme catalyzed an increase in the saccharification of Phragmites communis (reeds) powder besides bearing potential for conversion of biomass into biofuel production. Gong et al. (2013) screened and characterized a GH10 family of xylanase from an uncultured microorganism inhabiting cow rumen through functional analysis of a metagenomic (BAC) library. An extensive pH profile, greater pH stability, specifically at basic pH, increased substrate specificity with a lack of cellulase activity owes a versatile industrial application of this enzyme.

8.5.1.2 Proteases Proteases occupy a key position among biocatalysts due to their wide physiological roles linked to versatile commercial applications. Their presence is inevitable in industrial biotechnology, particularly in detergent, pharmaceutical, and food technology. Proteases constitute a large family of omnipresent enzymes found in a wide variety of living organisms, including microorganisms, plants, and animals. However, proteases from microbial origins show distinct advantages compared to plant or animal proteases, because they reveal almost all the characteristics required for biotechnological applications. In recent times, a large number of proteases have been discovered using metagenomics. Metagenomic DNA isolated from the goatskin surface revealed an overexpression of alkaline Serine protease (AS-protease) with a purified molecular mass of w63 kDa but was found inactive due to the formation of inclusion bodies. A novel mesophilic protease was derived from a metagenomic library constructed from Antarctic coastal sediment. Since the enzyme activity was inhibited by phenylmethylsulfonyl fluoride (PMSF, 1 mM) and AEBSF (hydrochloride 4-(2-aminoethyl)-benzenesulfonyl fluoride), it was assigned as a serine protease. The purified enzyme was found oxidant stable, thereby realizing its application in both detergent and bleaching industries. Two serine proteases were isolated from metagenomic libraries of the deserts of Gobi and Death Valley and a gene encoding alkaline protease was extracted from the saline habitat therein, the sequence analyses of which revealed it as serine proteases (Neveu et al., 2011).

8.5.1.3 Lipases Lipases, also called as triacylglycerol acylhydrolases (EC 3.1.1.3), are known to biocatalyze the hydrolysis of triaclyglycerol into glycerol and fatty acids. Being resistant to extreme conditions (high temperature, pH, presence of organic solvents, etc.), they have captured special industrial appeal. Lipases occur naturally in a variety of animal and plant species; however, the enzymes from microbial sources like bacteria, yeast, and fungi receive particular attention due to their real and potential applications in a vast range of industries mainly such as detergents, dairy, fats, and oils, as well as pharmaceutical industries. LipC12 (a Lipase-producing clone) reported of having high specific activity against long-chain triacylglycerols, a wide range of activity and stability over different pH and temperature values, solvent stability, and tolerance to high salt concentrations was isolated using a fat-contaminated soil exposed to an enrichment of prokaryotic DNA following a three-step screening strategy. Lipases have been reported from different metagenome ranging from thermal environments to saline lakes and from field soil to marine sediments, as well as drinking water (Sharma et al., 2001). Three novel genes having lipolytic and one gene having proteolytic activity were screened through mining a metagenome from volcanic thermal spring. A large number of lipolytic enzymes have been explored from coastal environments in marine sediments, including a lipase from the Baltic Sea and two esterases from the Arctic coast. EstAT11 is one of the esterases, which was found to hydrolyze (S)-racemic ofloxacin butyl ester having 70.3%, excess enantiomeric value, thereby showing its potential for heat-labile substrates. Lipo1 is a newly named esterase having unique characteristics. It was isolated from a metagenomic library screened from activated sludge. Lipo1 was optimally active at 10
C temperature and was resistant in the presence of detergents, making it useful as an additive in the laundry industry and organic chemistry as well. Alkaline tolerant esterases have been reported from compost soil metagenome but displayed a lack of thermostability. EstATII, an unusual esterase reported from Red Sea Atlantis II brine pool isolated using a function-based metagenomic approach was found to be thermophilic, displaying optimum activity at 65
C, halotolerant-maintaining average activity up to 4.5M NaCl and metal tolerant- 60% residual activity in the presence of different heavy metals. The abovementioned properties of the Red Sea Atlantis II brine pool esterase, make it a wide spectrum of the potential biocatalyst. From the metagenomic library of deep-sea sediments, six different lipolytic enzyme-encoding genes showing a low percentage of sequence similarity (approximately 33%e58% identity) to the existing proteins were isolated (Lee et al., 2015).

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8.5.1.4 Amylases Amylases, the well-known starch degrading enzymes are widely distributed across the microbial, plant, as well as animal kingdoms. Amylases are widely used in different industrial processes like fermentation, food, and pharmaceutical industries since they catalyze the hydrolysis of starch. AmyI3C6, a cold-adapted alpha-amylase, isolated from a metagenomic library of alkaline and cold environment postpurification, showed similarities to class Clostridia-a-amylases. When tested against two commercial detergents, the enzyme was found to display significant activity in both of them, thereby suggesting the potential use of AmyI3C6 a-amylase as a detergent additive, particularly in low-temperature laundry cleaning processes. A novel amylolytic enzyme with an ability to hydrolyze soluble starch, as well as cyclodextrins to yield high titers of maltose and catalyze pullulan to panose hydrolysis had genes encoded from a Metagenomic Library derived from the soil. The enzyme marked exclusive a-(1, 4) linkages showing high transglycosylation activity. The transglycosylation and hydrolytic properties of AmyM display that it is a novel enzyme that can be considered as a maltogenic amylase having intermediate properties of a-amylase, and 4-a-glucanotransferase. A novel amylase was discovered from a soil metagenome, which retained 90% of activity at an extremely low temperature making it a potential candidate for versatile industrial applications. A calcium-dependent thermostable amylase isolated from a soil metagenome was shown to have applications in the baking industry (Souza et al., 2010).

8.5.1.5 Cellulases Cellulases refers to the class glycosyl hydrolases that contains three specific types of enzymatic activities viz., (i) endoglucanases (EC 3.2.1.4), (ii) exoglucanases and (iii) b-glucosidases (EC 3.2.1.21). Cellulases have drawn more interest due to their diverse applications. Besides their use in animal feeds as enhancers of quality and digestibility of nutrients, they find an important role in fruit juice processing, baking, and deinking of paper. Cellulases are known to be isolated from diverse natural environments such as soil, compost, rumen, etc., using metagenomic techniques through the construction of metagenomic libraries and subsequent screening of the clones that are biologically active. Cellulase enzymes have also been reported from niche environments such as anaerobic digester, saline, and alkaline lakes, etc., Yeh and his coworkers in 2013 reported a cellulase gene-GH12, RSC-EG1, which was found to encode 464 amino acids and two other ORFs (Open Reading Frames), which were isolated from metagenomic library obtained from rice straw compost showing more than 70% similarity at the level of amino acid with cellulase derived from Thermobispora sp and Micromonospora aurantiaca (Zhang et al., 2013). Among the microbial consortium sampled from forest soil, rotted tree, elephant dung, and cow rumen, seven independent clones were isolated and identified showing diverse cellulase activities (05-endo-b-1,4-glucanases and 02- b-glucosidases), with less than 50% identities and about 70% similarities to cellulases existing in the databases. Yadan and his coworkers reported unglu135B12-a b-glucosidase gene, belonging to Glycoside Hydrolase family 3. In 2013 a novel CelEx-BR12 gene was identified and characterized by Kyong and his coworkers, having ORF of 1140 base pairs encoding a 380-amino-acid-protein (41.8 kDa) from rumen bacteria employing a highthroughput robotic screening system. The enzyme was found to be multifunctional showing activities against fluorogenic and natural glycosides, such as CMC (105.9 U/mg, 4-methylumbelliferyl-b- d-cellobioside (0.3 U mg), oat spelt xylan (67.9 U/mg) birchwood xylan (132.3 U/mg), and 2-hydroxyethyl-cellulose (26.3 U/mg) relating their industrial use. A metagenome-derived halotolerant cellulase was also characterized, showing a high degree of stability, unveiling the significance of metagenomics cellulases (Voget et al., 2003).

8.5.2 Metagenomics and pharmaceuticals The latter half of the 20th century has witnessed several antibiotics and other medical agents isolated from microbes showing dramatic improvement in human health. The traditional culturing method for the isolation and screening of microorganisms has resulted in the rediscovery of some well-known antibiotics (99%). Hence, a novel approach is required for the discovery of new drugs. One of the novel approaches that may prove the rich sources of new antibiotics happens to be metagenomics. Antibiotics Turbomycin A and B were isolated from Metagenomic Library derived from soil microbial DNA (Gillespie et al., 2002). Novel Marine microbial natural products (MMNPs) were discovered following routine screenings, and adopting strategies, including metagenomics, genomics, followed by combinatorial biosynthesis along with synthetic biology (Xiong et al., 2013). More than 30 biochemical compounds obtained from marine microorganisms like didemnin B and thiocoraline are undergoing clinical and/or preclinical studies to resolve the treatment of various types of cancers. The gene for indirubin production was reported from a soil metagenome, which, besides producing red indirubin pigment, displayed antibacterial activity (MacNeil et al., 2001). Lately, the antibacterial activity was found to be an effect of indirubin along with an unknown unpigmented component. In another study, soil DNA was extracted, purified,

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and partially digested, and the fragments obtained were inserted into a suitable vector (Escherichia coli) for expression. Resulted clones were found to express both enzymatic and antibiotic activities being encoded by novel sequences (Robin).

8.5.3 Metagenomics and biosurfactants Biosurfactants have found an advantage over chemical surfactants being nontoxic and easy degradation in the environment. The chemical surfactants, on the other hand, are toxic and cannot be readily degraded. Greater environmental concerns, advancements in biotechnology along with the emergence of stringent laws have altogether led to the search for a potential alternative to the widespread chemical surfactants available in the market. Biosurfactants have proved to be potential replacements for synthetic surfactants in several applications like food, pharmaceutical, and biomedical industry, industrial processes, which include wetting, lubrication, softening, making emulsions, fixing dyes, foaming, antifoaming, stabilizing dispersions, and bioremediation of inorganic and organic contaminated sites. DNA libraries have been constructed from the petroleum-contaminated samples (such as water, soil, etc.) using metagenomics subsequently screened for biosurfactant producing clones. Two bacteria (A-1 and B-1) were reported by Morikawa et al. (1992), exhibiting large halos emulsified on oil-L-agar plates around the colonies. Both bacteria were found to produce the same biosurfactant-surfactin. Biosurfactants can be isolated from metagenomics libraries using function-based approaches that include SIGEX (substrateinduced gene expression) (Bashir et al., 2014) and HTP (High throughput) screening (Saptute et al., 2010). Functional screens have been described for the isolation of biosurfactants, which along with different approaches, can be employed to overcome some typical problems generally encountered while performing functional metagenomic-based screens (Kennedy et al., 2011).

8.5.4 Metagenomics and biodegradation Wastes generated through petroleum spills, incomplete combustion of fossil fuels, etc., by industries have led to an accumulation of petroleum hydrocarbons in the environment. Such anthropogenic compounds introduce large amounts of aromatic hydrocarbons into the environment annually, thereby contaminating ecosystems. Microorganisms being straightly involved in biogeochemical cycles drive degradation of several carbon sources, along with petroleum hydrocarbons, through the break down of aromatic rings, such as those present in xylene, toluene, and benzene, thereby mineralizing the carbon skeleton. Here, metagenomics could be used as a tool that can eliminate steps related to cultivation, since it involves direct extraction of DNA from the environment and its cloning in a suitable vector. The potential of the metagenomic approach has been reported for identification and elucidation of novel genes and pathways in less studied environments, which contribute toward a broader perspective in relation to the hydrocarbon degradation processes, especially in petroleum reservoirs (Knapik et al., 2019). A function-driven metagenomic approach has been opted to decipher novel functional metabolic pathways involved in the biodegradation of aromatic compounds from a metagenomic library derived from an oil reservoir. Researchers have identified metabolic pathways and genes related to the degradation of aromatic compounds and phenol present in sludge samples of a petroleum refinery wastewater, using a metagenomic approach to screen a broader range of functional diversity. Bacterial populations have been reported in cold marine ecosystems with an ability to degrade polycyclic aromatic hydrocarbon (PAH) through the identification of functional targets (Marcos et al., 2009). In the study, 14 specific groups of genes have been reported, most of which show a significant relationship with dioxygenases enzymes from Gram-positive bacteria belonging to genera Bacillus, Rhodococcus, Mycobacterium, Terrabacter, and Nocardioides. These results are indicative of the presence of substantial diversity of unidentified dioxygenases genes, especially in the cold, polluted environment. The above-said information could be employed as a start point for the design of quantitative molecular tools for analyzing the abundance and dynamics of aromatic hydrocarbon-degrading bacterial populations, particularly in the marine environment.

8.6 Conclusions and future perspectives Metagenomics provides a directed approach to study exotic habitats, design culture media for previously-uncultured microbes, examine genes predominating in a given environment, understand metabolic pathways and to examine and understand the diversity patterns of microorganisms or phylogenetic diversity using 16S rRNA that can be used to monitor, assess and predict environmental conditions and any changes therein. It further elucidates the examination of genes and/or operons as desirable enzymatic candidates such as lipases, cellulases, chitinases, amylases, antibiotics, and other natural products that may be exploited for diverse industrial and pharmaceutical applications. It helps in examining signal transduction mechanisms associated with samples or secretory and regulatory roles of genes of interest. Bacteriophage or

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plasmid sequences potentially influence the diversity and structure of microbial communities. Metagenomics helps in examining such potential lateral gene transfer events besides providing information on genome plasticity, which may be useful in hypothesizing a multitude of selective pressures existing for gene capture and its further evolution within a habitat. Altogether, metagenomic metadata and data can be directed to design both low- and high-throughput experiments that can unveil the roles of microorganisms and genes toward the establishment of a dynamic microbial community. Since metagenomics can only progress with the progress of library and sequencing technologies, an improvement in bioinformatics can ease the analysis of library profiling besides quickening the process.

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Glenn, T., 2014. NGS Field Guide: OverviewjThe Molecular Ecologist. Available online at: http://www.molecularecologist.com/next-gen-fieldguide2014/. Gong, X., Gruniniger, R.J., Forster, R.J., Teather, R.M., McAllister, T.A., 2013. Biochemical analysis of a highly specific, pH stable xylanase gene identified from a bovine rumen-derived metagenomic library. Appl. Microbiol. Biotechnol. 97 (6), 2423e2431. Handelsman, J., 2004. Metagenomics: application of genomics to uncultured microorganisms. Microbiol. Mol. Biol. Rev. 68 (4), 669e685. Kariin, S., Burge, C., 1995. Dinucleotide relative abundance extremes: a genomic signature. Trends Genet. 11 (7), 283e290. Kasianowicz, J.J., Brandin, E., Branton, D., Deamer, D.W., 1996. Characterization of individual polynucleotide molecules using a membrane channel. Proc. Natl. Acad. Sci. U.S.A. 93 (24), 13770e13773. Kennedy, J., O’Leary, N.D., Kiran, G.S., Morrissey, J.P., O’Gara, F., Selvin, J., Dobson, A.D.W., 2011. Functional metagenomic strategies for the discovery of novel enzymes and biosurfactants with biotechnological applications from marine ecosystems. J. Appl. Microbiol. 111 (4), 787e799. Knapik, K., Becerra, M., González-Siso, M.-I., 2019. Microbial diversity analysis and screening for novel xylanase enzymes from the sediment of the Lobios Hot Spring in Spain. Sci. Rep. 9 (1), 11195. Knietsch, A., Waschkowitz, T., Bowien, S., Henne, A., Daniel, R., 2003. Construction and screening of metagenomic libraries derived from enrichment cultures: generation of a gene bank for genes conferring alcohol oxidoreductase activity on Escherichia coli. Appl. Environ. Microbiol. 69 (3), 1408e1416. Kultima, J.R., Sunagawa, S., Li, J., Chen, W., Chen, H., Mende, D.R., Arumugam, M., Pan, Q., Liu, B., Qin, J., Wang, J., Bork, P., 2012. MOCAT: a metagenomics assembly and gene prediction toolkit. PLoS One 7 (10), e47656. Lee, L.P., Karbul, H.M., Citartan, M., Gopinath, S.C.B., Lakshmipriya, T., Tang, T.-H., 2015. Lipase-secreting Bacillus species in an oil-contaminated habitat: promising strains to alleviate oil pollution. BioMed Res. Int. 2015, 1e9. Logares, R., Haverkamp, T.H.A., Kumar, S., Lanzén, A., Nederbragt, A.J., Quince, C., et al., 2012. Environmental microbiology through the lens of highthroughput DNA sequencing: synopsis of current platforms and bioinformatics approaches. J. Microbiol. Methods 91 (1), 106e113. Luo, C., Tsementzi, D., Kyrpides, N., Read, T., Konstantinidis, K.T., 2012. Direct comparisons of Illumina vs. Roche 454 sequencing technologies on the same microbial community DNA sample. PLoS One 7 (2), e30087.

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Nelson, M.T., Pope, C.E., Marsh, R.L., Wolter, D.J., Weiss, E.J., Hager, K.R., Vo, A.T., Brittnacher, M.J., Radey, M.C., Hayden, H.S., Eng, A., Miller, S.I., Borenstein, E., Hoffman, L.R., 2019. Human and extracellular DNA depletion for metagenomic analysis of complex clinical infection samples yields optimized viable microbiome profiles. Cell Rep. 26 (8), 2227e2240 e5. Neveu, J., Regeard, C., DuBow, M.S., 2011. Isolation and characterization of two serine proteases from metagenomic libraries of the Gobi and Death Valley deserts. Appl. Microbiol. Biotechnol. 91 (3), 635e644. Ngara, T.R., Zhang, H., 2018. Recent advances in function-based metagenomic screening genomics, proteomics and bioinformatics. Dev. Reprod. Biol. 16 (6), 405e415. Østergaard, L., Yanofsky, M.F., 2004. Establishing gene function by mutagenesis in Arabidopsis thaliana. Plant J. 39 (5), 682e696. Parages, M., Gutiérrez-Barranquero, J., Reen, F., Dobson, A., O’Gara, F., 2016. Integrated (meta) genomic and synthetic biology approaches to develop new biocatalysts. Mar. Drugs 14 (3), 62. Poretsky, R., Rodriguez-R, L.M., Luo, C., Tsementzi, D., Konstantinidis, K.T., 2014. Strengths and limitations of 16S rRNA gene amplicon sequencing in revealing temporal microbial community dynamics. PLoS One 9 (4), e93827. Quast, C., Pruesse, E., Yilmaz, P., Gerken, J., Schweer, T., Yarza, P., Peplies, J., Glöckner, F.O., 2013. The SILVA ribosomal RNA gene database project: improved data processing and web-based tools. Nucleic Acids Res. 41, D590eD596. Ramya, S.B., Sowjanya, B., Divakar, K., 2019. Metagenomic bioprospecting of novel oxygen insensitive nitroreductase for degradation of nitro aromatic compounds. Int. Biodeterior. Biodegrad. 143, 104737. Riesenfeld, C.S., Schloss, P.D., Handelsman, J., 2004. Metagenomics: genomic analysis of microbial communities. Annu. Rev. Genet. 38 (1), 525e552. Rocha-Martin, J., Harrington, C., Dobson, A., O’Gara, F., 2014. Emerging strategies and integrated systems microbiology technologies for biodiscovery of marine bioactive compounds. Mar. Drugs 12 (6), 3516e3559. Rothberg, Jonathan, M., Wolfgang, H., Todd, M.R., Jonathan, S., William, M., Mel, D., John, H.L., et al., 2011. An integrated semiconductor device enabling non-optical genome sequencing. Nature 475 (7356), 348e352. Sanchez-Flores, A., Abreu-Goodger, C., 2014. A practical guide to sequencing genomes and transcriptomes. Curr. Top. Med. Chem. 14 (3), 398e406. Satpute, S.K., Banat, I.M., Dhakephalkar, P.K., Banpurkar, A.G., Chopade, B.A., 2010. Biosurfactants, bioemulsifiers and exopolysaccharides from marine microorganisms. Biotechnol. Adv. 28 (4), 436e450. Schmidt, T.M., DeLong, E.F., Pac, N.R., 1991. Analysis of a marine picoplankton community by 16S rRNA gene cloning and sequencing. J. Bacteriol. 173 (14), 4371e4378. Sharma, R., Chisti, Y., Banerjee, U.C., 2001. Production, purification, characterization, and applications of lipases. Biotechnol. Adv. 19 (8), 627e662. Sharpton, T.J., 2014. An introduction to the analysis of shotgun metagenomic data. Front. Plant Sci. 5, 209. Simon, C., Daniel, R., 2010. Metagenomic analyses: past and future trends. Appl. Environ. Microbiol. 77 (4), 1153e1161. Sousa de STP, Cabral, L., Lacerda-Júnior, G.V., Noronha, M.F., Ottoni, J.R., Sartoratto, A., de Oliveira, V.M., 2019. Exploring the genetic potential of a fosmid metagenomic library from an oil-impacted mangrove sediment for metabolism of aromatic compounds. Ecotox. Environ. Safe 189, 109974. Souza, P.M., Magalhães, P.O., 2010. Application of microbial a-amylase in industry - a review. Braz. J. Microbiol. 41 (4), 850e861. Steele, H.L., Jaeger, K.E., Daniel, R., Streit, W.R., 2009. Advances in recovery of novel biocatalysts from metagenomes. J. Mol. Microbiol. Biotechnol. 16 (1e2), 25e37. Su, X., Pan, W., Song, B., Xu, J., Ning, K., 2014. 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Chapter 9

Molecular markers and its application in animal breeding Reshma Raj S1 and D.N. Das2 1

ICAR-National Dairy Research Institute, Bangalore, Karnataka, India; 2Genetics Laboratory, Dairy Production Section, ICAR-National Dairy

Research Institute (SRS), Southern Regional Station, Bangalore, Karnataka, India

9.1 Introduction Molecular markers are defined as any stable and inherited variation, which is quantifiable or detectable by a suitable method and can be subsequently used to detect the presence of a specific genotype or phenotype. Molecular markers provide useful information about allelic variation at a given locus and are a powerful aid to animal breeding that defines the genetic makeup (genotype) and predicts the performance of the animal. Marker assisted selection (MAS) is one of the approaches that assist in animal breeding. MAS is used for the improvement of a desirable trait by utilizing the genetic diversity in the breeding population. The methods used to define molecular markers include Restriction Fragment Length Polymorphisms (RFLPs), Random Amplified Polymorphic DNA (RAPD), Amplified Fragment Length Polymorphism (AFLP), Microsatellites, Minisatelites, Single Nucleotide Polymorphisms (SNPs) and many more. Linkage analysis, association analysis, and analysis of gene function can be used to determine the polymorphisms that are useful markers for desirable traits. Many important traits, such as growth rate, milk production, wool production, and litter size are multifactorial, that is they are controlled by both genes and environment. The increasing availability of molecular markers in livestock enables the analyses and evaluation of genetic diversity and the detection of genes influencing economically important traits. In recent times molecular genetics has made a little direct contribution to animal breeding by understanding gene structure and expression with the help of various marker techniques.

9.2 Quantitative and molecular genetics Quantitative genetics is the study of the genetic basis underlying phenotypic variation among individuals. Quantitative traits are polygenic, i.e., they are controlled by many genes, and there are environmental effects that alter the phenotypic state of each individual. The value of quantitative traits varies continuously. Examples include milk production, height, weight, and longevity. Therefore, the genetics of continuously varying characters is known as quantitative genetics. Instead of considering changes in the frequencies of specific alleles of genotypes, quantitative genetics quantify changes in the frequency distribution of traits, which cannot be easily placed in discrete phenotypic classes. Heritability (H2) in “broad sense” is defined as the proportion of total phenotypic variation that is attributable to genetic factors. It measures the relative importance of genes in determining phenotypic variance. Heritability (h2) in “narrow sense” is the proportion of total phenotypic variation that is due to additive genetic variance. It is important to identify the response of a population to natural or artificial selection. Prediction of responses to selective animal breeding and understanding evolution in natural population relies on the awareness of the factors underlying variation in quantitative traits. Quantitative genetic variation was studied traditionally with no prior knowledge about the genes involved using statistical analyses to partition components of phenotypic variation. However, ongoing developments in genomic technology lead to increasing possibility of identifying specific genes that underlie variation in quantitative traits. Molecular genetics is a branch of genetics, which deals with the structure and activity of genetic material at the molecular level. The field of study is based on merging of various subfields in biology viz. cellular biology, classical Mendelian inheritance, molecular biology, biochemistry, and biotechnology. The molecule responsible for heredity was

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identified through the discovery of DNA as a means to transfer the genetic code of life from one cell to another and between generations. The discovery of the structure of DNA by Watson and Crick, along with Franklin and Wilkins, was a milestone for molecular genetics. Isolation of a restriction endonuclease in E. coli by Arber and Linn in 1969 opened the field of genetic engineering. Restriction enzymes are used to cut DNA into smaller fragments at specific nucleotide sequences and linearize DNA for electrophoretic separation. In 1971, Berg utilized restriction enzymes to create the first recombinant DNA molecule and the first recombinant DNA plasmid. In 1972, Cohen and Boyer created the first recombinant DNA organism by inserting recombinant DNA plasmids into E. coli, now known as bacterial transformation, and paved the way for molecular cloning. The development of DNA sequencing techniques in the late 1970s, first by Maxam and Gilbert, and then by Frederick Sanger, was pivotal to molecular genetic research and enabled scientists to begin conducting genetic screens to relate genotypic sequences to phenotypes. Polymerase chain reaction (PCR) using Taq polymerase, invented by Mullis in 1985, enabled scientists to create millions of copies of a specific DNA sequence that could be used for transformation or manipulated using agarose gel separation. A decade later, the first whole genome was sequenced (Haemophilus influenzae), followed by the eventual sequencing of the human genome via the Human Genome Project in 2001. Genetic variation among important traits in livestock can be explained by the infinitesimal model and the finite loci model. The infinitesimal model is the basis of quantitative genetics, and the finite loci model is the basis of molecular genetics. The infinitesimal model assumes that traits are determined by an infinite number of unlinked and additive loci, each with an infinitesimally small effect (Fischer, 1918). This model is valuable for animal breeding and forms the basis for breeding value estimation theory (Henderson, 1984). The finite loci model assumes the existence of a finite amount of genetically inherited material. Many instances of evidence confirmed that the distribution of the effect of these loci on quantitative traits could be classified into a few genes with large effects and many with small effects (Shrimpton and Robertson, 1988).

9.3 Molecular markers Marker is a piece of DNA molecule that is associated with a certain trait of an organism. Types of markers include Morphological, Biochemical, Chromosomal, and Genetic. Morphological markers use external animal characteristics as a marker (coat colour, skin structure, udder shape, body shape, and anatomical characteristics) (Van Wezel and Rodgers, 1996). Biochemical markers represent biochemical traits that can be analyzed by protein electrophoresis (blood type and isozymes). A genetic marker is a gene or DNA sequence with a known location on a chromosome and is associated with a specific gene or trait. It can be described as a variation, which may arise due to mutation or alteration in the genomic loci that can be observed. A genetic marker may be a short DNA sequence, like a sequence surrounding a single base pair change (single nucleotide polymorphism, SNP), or a long one, such as minisatellite and microsatellite. Molecular markers are classified into two categories: type I markers that are associated with genes of known function, and type II markers that are associated with anonymous genomic segments (O’Brien, 1991). Genetic markers can also be classified based on chronology into three different categories: “First Generation Markers” (RFLP, RAPD and their modifications), “Second Generation Markers” (AFLP, SSRs, and their modifications), and “New Generation Markers” (ESTs and SNPs) (Maheswaran, 2004). These markers help in the identification of dominance and codominance within the genome. Dominance and codominance identification with a marker help to differentiate heterozygotes from homozygotes within the organism. Codominant markers are more useful since they can identify more than one allele and allows following a particular trait through mapping techniques. Molecular markers allow amplification of specific sequences within the genome, and help in comparison and analysis. Molecular markers can also be classified as Hybridization-based Markers (RFLP), PCR-based Markers (RAPD, AFLP, Microsatellites), and DNA Chip and Sequencing-based Markers (SNPs). Molecular markers give more accurate genetic information and a better understanding of the animal genetic resources. Molecular markers are effective in the following ways: l

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Abundance of genetic linkage between identifiable locations within a chromosome is identified and can be repeated for verification. Small changes within the mapping population can be identified and allow distinction between a mapping species, for segregation of traits and identity. Identification of particular locations on a chromosome and enable the creation of physical maps. Identifying how many alleles an organism has for a particular trait (biallelic or polyallelic) (Maheswaran, 2004).

Application of molecular marker for genetic improvement depends upon the ability to genotype individuals for specific genetic loci. For these purposes, three types of observable polymorphic genetic loci are generally chosen (Dekkers, 2004),

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1. Direct markers: loci that code for the functional mutation. 2. LD markers: loci that are in population-wide linkage disequilibrium with the functional mutation. 3. LE markers: loci that are in population-wide linkage equilibrium with the functional mutation in outbred populations. These marker loci differ not only in the method of detection but also in their application in selection programs. While direct markers and to a lesser degree, LD markers, allow selection on genotype across the population due to the consistent association between genotype with phenotype, use of LE markers generally permit different linkage phases between markers and QTL from family to family. Direct markers are preferred for effective implementation of MAS, followed by LD and LE markers, the latter requiring within-family analysis and selection. Ease of application and potential for extra genetic gain is greatest for direct markers, followed by LD markers, but is antagonistic to ease of detection, which is greatest for LE markers. Thus, the ease and ability to use markers in selection is opposite to their ease of detection and increases from direct markers to LD markers and LE markers. Selection on these three types of markers is referred to as gene assisted selection (GAS), LD marker assisted selection (LD-MAS), and LE marker assisted selection (LE-MAS).

9.3.1 Restriction fragment length polymorphism (RFLP) Restriction fragment length polymorphism is a technique invented by Alec Jeffreys in the year 1984. RFLP is a technique that allows the exploitation of variations in homologous DNA sequences, which is known as polymorphisms. It is used to differentiate individuals, populations, or species, and also helps to locate genes within a sequence. RFLPs are bands corresponding to DNA fragments obtained by the digestion of genomic DNA with restriction endonucleases (size: 2e10 kb). Yang et al. (2013) reported that nucleotide base substitutions, deletions, insertions, inversions, and duplications within the whole genome can remove or create new restriction sites. RFLP analysis was the first DNA profiling technique with widespread application. RFLP analysis was an important tool in the localization of genes for genetic disorders, genome mapping, determination of risk for disease, and paternity testing.

9.3.1.1 Principle of RFLP RFLP is an enzymatic procedure used for the separation and identification of desired fragments of DNA. Restriction enzymes are used to obtain desired DNA fragments and are detected by Southern hybridization.

9.3.1.2 RFLP technique The basic technique for detecting RFLPs includes fragmentation of a sample of DNA with a restriction endonuclease. Restriction endonucleases are enzymes that cut lengthy DNA into short fragments. Each restriction endonuclease targets different nucleotide sequences in a DNA strand, and therefore, cuts at different sites. The distance between the cleavage sites of restriction endonuclease differs between individuals. Therefore, the length of the DNA fragments obtained by a restriction endonuclease will vary across both individual organisms and species. Restriction enzymes selectively cleave a DNA molecule wherever a short, specific sequence is recognized in a process known as a restriction digest. The recognition sites of these enzymes are generally four to six base pairs in length. The shorter the sequence recognized, the greater the number of fragments generated from digestion. DNA fragments are separated by agarose gel electrophoresis based on their charge and size. The fragmented DNA samples are loaded in the wells of agarose gel placed in the electrophoretic unit containing the buffer and the anodecathode electrodes. When an electric field is applied, the negatively charged DNA fragments migrate toward the positive electrode. Smaller fragments will move faster through the gel, and the larger ones are left behind, thus separating the DNA samples into distinct bands on the gel based on the size of fragments. Southern blot hybridization is subsequently used to detect the fragments by labeled DNA probe (Fig. 9.1). Radioactive isotope or nonradioactive stains like fluorescein or digoxigenin is used for probe labeling. RFLP probe is locus-specific and comprises a homologous sequence of a particular chromosomal region. Probes are generated by constructing genomic or complementary DNA (cDNA) libraries, and consist of a specific sequence of unknown identity (genomic DNA) or part of the sequence of a functional gene (exons only, cDNA). RFLP probes are maintained as clones in the appropriate bacterial vectors, which enable the isolation of DNA fragments they hold. Heterologous probes can also be used (probes from related species). DNA sequence variation influencing the presence or absence of recognition sites of restriction enzymes, and insertions and deletions within two adjacent restriction sites, form the basis of restriction fragment length polymorphisms.

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FIGURE 9.1 RFLP technique.

9.3.1.3 Applications of RFLP RFLP technique was used for early methods of genetic fingerprinting, identification of samples retrieved from crime scenes, paternity determination, and characterization of genetic diversity or breeding patterns in animal populations. RFLPs can also be applied in diversity and phylogenetic studies ranging from individuals within populations or species to closely related species. Emadi et al. (2010) stated that the analysis of RFLP variation in genomes was an important tool for genetic disease analysis and genome mapping. The chromosomal location of a specific disease gene is determined by the analyses of DNA of family members afflicted by the disease and checked for RFLP alleles that show a pattern of inheritance similar to that of the disease. Once a disease gene is localized, RFLP analysis of other families could reveal who is at risk for the disease, or who is likely to be a carrier of the mutant genes. RFLPs have been widely used in gene mapping studies because of their high genomic abundance due to the ample availability of different restriction enzymes and random distribution throughout the genome. It is used for the analysis of unique patterns in DNA fragments for genetically differentiating between organisms.

9.3.1.4 Limitations of RFLP l l l

RFLP technique is slow and cumbersome. It requires a large quantity of sample DNA. The combined process of probe labeling, DNA fragmentation, electrophoresis, blotting, hybridization, washing, and autoradiography can take up to a month to complete.

Saiki et al. (1985) reported another version of the RFLP technique, which utilizes oligonucleotide probes. Human Genome Project has replaced the need for RFLP mapping to an extent. Identification of several SNPs in the human genome project (as well as the direct identification of many disease genes and mutations) has replaced the need for RFLP in disease linkage analysis. However, RFLP is still used in MAS. Terminal restriction fragment length polymorphism (TRFLP or T-RFLP) is a technique developed for the characterization of bacterial communities in mixed-species samples. TRFLP technique works by PCR amplification of genomic DNA using primer pairs labeled with fluorescent tags. PCR products are digested using restriction enzymes, and the patterns are visualized using a DNA sequencer. Analyses are done either by simply counting and comparing bands or peaks in the TRFLP profile or by matching bands from one or more TRFLP runs to a database of known species. The technique is similar to temperature gradient or denaturing gradient gel electrophoresis (TGGE and DGGE).

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9.3.2 Random amplified polymorphic DNA (RAPD) RAPD is a PCR-based technique used for the identification of genetic variation. The technique utilizes a single random primer in a PCR reaction, which results in the amplification of several distinct DNA products. RAPD technique was developed independently by two different laboratories and called as RAPD (Williams et al., 1990) and AP-PCR (Arbitrarily primed PCR) (Welsh and McClelland, 1990). Kumar and Gurusubramanian (2011) reported that the PCR-based RAPD technique is one of the most commonly used molecular techniques in the last decade to develop DNA markers. In this reaction, a single primer of arbitrary nucleotide sequence binds at two different sites on opposite strands of the DNA template. The PCR amplification results in distinct DNA products if these priming sites are within an amplifiable distance of each other. Differences in sequence in one or both the primer binding sites results in polymorphisms between individuals. These polymorphisms are visible as the presence or absence of a specific RAPD band, and such polymorphisms behave as dominant genetic markers. RAPD technology provides a quick and efficient screen for DNA sequence-based polymorphism at a very large number of loci. RAPD technique does not require the presequencing of DNA (Nandani and Thakur, 2014). Weising et al. (2005) reported that RAPDs are technically simple and independent of any prior DNA sequence information. Lynch and Milligan (1994) opined that RAPDs are advantages over RFLP and fingerprint. Only low quantities of template DNA are required for RAPD because of the involvement of PCR. Primer designing does not require any sequence data as random primers are commercially available.

9.3.2.1 Principle of RAPD The principle of RAPD is that a single, short oligonucleotide primer, which binds to many different loci, is used for amplifying random sequences from a complex DNA template. This means that the amplified fragment generated by PCR depends on the length and size of both the primer and the target genome (Nandani and Thakur, 2014).

9.3.2.2 RAPD technique A target DNA sequence is amplified exponentially with the help of a thermostable DNA polymerase, random primers, dideoxynucleotide triphosphates (ddNTPs), magnesium and reaction buffer. The reaction involves repeated cycles, each cycle comprising of several steps, including denaturation, annealing, and extension. In the first step, i.e., denaturation, the double-stranded DNA (dsDNA) is made into single-stranded DNA (ssDNA) by increasing the temperature to 94
C. In the second step, the temperature is lowered to about 40e65
C resulting in annealing of the primer to their target sequences on the template DNA (annealing step). The third step is elongation/extension, where the thermostable Taq DNA polymerase extends the 30 ends of the DNA-primer hybrids toward the other primer binding site. Here the temperature is set to 72
C, and the activity of thermostable Taq DNA polymerase is optimal at this temperature. This happens at both primerannealing sites on both the DNA strands, and thus, the target fragment is replicated completely. These three steps are repeated 40 to 50 times resulting in the exponential amplification of the target between the 50 ends of the two primer binding sites. Amplification products are separated by agarose gel electrophoresis and visualized by staining with ethidium bromide. The factors that need to be optimized in RAPD reaction include enzyme concentration, magnesium concentration, the annealing temperature of the primer, and DNA concentration. The G þ C content of the primers should be 40%e60%, and care should be taken to avoid sequences that produce internal secondary structures.

9.3.2.3 Applications of RAPD RAPDs can be used for studies ranging from the individual level (e.g., genetic identity) to closely related species. RAPD technique is used in gene mapping studies to fill gaps not covered by other markers. Arbitrarily Primed Polymerase Chain Reaction (AP-PCR), a variant of RAPD technique, uses longer arbitrary primers than RAPDs. Another variant, DNA Amplification Fingerprinting (DAF), uses shorter primers (five to eight bp) to generate a larger number of fragments.

9.3.2.4 Limitations of RAPD l

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RAPDs have low reproducibility; therefore, highly standardized laboratory protocols are required because of their sensitivity to reaction conditions. The quality and concentration of template DNA, concentrations of PCR components, and the PCR cycling conditions greatly influence the outcome. So, purified and high molecular weight DNA is required for RAPD analyses.

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Polymorphism by RAPD technique is detected as the presence or absence of a band of a certain molecular weight, with no information on heterozygosity besides being dominantly inherited (Brumlop and Finckh, 2011). Nearly all RAPD markers are dominant; hence, it is not possible to differentiate whether a DNA segment is amplified from a locus that is heterozygous or homozygous. Codominant RAPD markers are observed as different-sized DNA segments amplified from the same locus and are rarely detected. Low reproducibility makes RAPDs unsuitable markers for transference or comparison of results in similar species and subjects. RAPD markers are not locus-specific, band profiles cannot be interpreted in terms of loci and alleles (dominance of markers), and similar sized fragments may not be homologous. RAPD results may be difficult to interpret, as mismatches between the primer and the template can lead to total absence or merely decreased amount of the PCR product.

9.3.3 Amplified fragment length polymorphism (AFLP) Amplified Fragment Length Polymorphism (AFLP) technique was developed by Keygene in the early 1990s. AFLP is a PCR-based technique, and it involves both RFLP and PCR. Multilocus DNA markers are generated by PCR without prior sequence information using cleaved genomic DNA as a template. The AFLP technique generates DNA fragments of size 80e500 bp from the digestion of genomic DNA with restriction enzymes, then by ligation of oligonucleotide adapters to the digestion products and selective amplification by PCR. The DNA fragments are separated and visualized by agarose gel electrophoresis. The AFLP banding profiles are the result of variations in the restriction sites or in the intervening region. AFLPs are considered as dominant markers. Advantages of AFLP include high genomic abundance, considerable reproducibility, high sensitivity, generation of many informative bands per reaction, and do not require sequence data for primer construction. AFLPs can be analyzed on automatic sequencers, but some systems are encountered with software problems concerning AFLP scoring.

9.3.3.1 Principle of AFLP AFLP analysis belongs to the category of selective restriction fragment amplification techniques, which are based on the ligation of adapters to genomic restriction fragments followed by a PCR-based amplification with adapter-specific primers (Vaneechoutte, 1996).

9.3.3.2 AFLP technique Only a small quantity of purified genomic DNA is required for the analysis of AFLP, and this genomic DNA is digested with two restriction enzymes, one with an average cutting frequency like EcoRI and other with a higher cutting frequency like MseI or TaqI. The fragments are ligated with site-specific recognition oligonucleotide adapters. A subset of approximately 50e100 restriction fragments is then selectively amplified in two consecutive PCRs under highly stringent conditions with adapter-specific primers. The amplified restriction fragments are radioactive or fluorescent-labeled and separated by agarose gel electrophoresis (Fig. 9.2). Selectivity is achieved by designing PCR primers that anneal specifically to the adaptor and the recognition site and carry one to three arbitrary chosen nucleotides at the 30 end. Only those restriction fragments will be amplified that have on both ends nucleotides complementary to the adapter and the recognition site plus the arbitrary nucleotide extension. The patterns obtained from different strains are polymorphic due to (i) mutations in the restriction sites, (ii) mutations in the sequences adjacent to the restriction sites and complementary to the selective primer extensions, and (iii) insertions or deletions within the amplified fragments.

9.3.3.3 Applications of AFLP Hedrick (1992) reported that widest applications of AFLP markers are found in the analyses of genetic variation below the species level, especially in the study of population structure and differentiation. AFLP technique produces highly replicable markers from DNA. AFLP technology is widely being used for the identification of genetic variation in strains or closely related species of bacteria, fungi, plants, and animals due to its ability to identify various polymorphisms in different genomic regions simultaneously and also due to its high sensitivity and reproducibility. AFLP technology is used in criminal investigation, paternity tests, determination of slight differences within populations, identification of clones and phylogenetic studies of closely related species, and in linkage studies to generate maps for quantitative trait loci (QTL)

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FIGURE 9.2 AFLP technique.

analysis. High genomic abundance and random distribution throughout the genome make AFLPs a widely valued technology for gene mapping studies. Moreover, AFLPs produce highly informative fingerprinting profiles. AFLPs are advantageous over RAPD, RFLP, and microsatellites. Mueller and Wolfenbarger (1999) reported that AFLP has the capacity of higher reproducibility, resolution, and sensitivity at the whole genome level as compared to other molecular techniques and also has the ability to amplify between 50 and 100 fragments at one time. Brumlop and Finckh (2011) stated that reproducibility, time and cost efficiency, and resolution of AFLPs are higher or equal as compared to RFLP, RAPD, and microsatellites. Meudt and Clarke (2007) suggested that amplification does not require prior sequence information. As a result, AFLP has become extremely beneficial in the study of taxa, including bacteria, fungi, plants, and animals. Ajmone-Marsan et al. (2002) opined that the AFLP method is an ideal molecular approach for population genetics and genome typing, and is widely applied for the detection of genetic polymorphisms, evaluation, and characterization of animal genetic resources.

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Purified, high molecular weight DNA is required for the AFLP technique. AFLP also requires the dominance of alleles and the possible nonhomology of comigrating fragments belonging to different loci. Certain strict criteria need to be adopted for the acceptance of bands in the analysis, because of the high number and different intensity of bands per primer combination.

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AFLP bands are not always independent. For example, in the case of insertion between two restriction sites, the amplified DNA fragment results in increased band size. This will be interpreted as the loss of a small band, and at the same time as the gain of a larger band. This is important for the analysis of genetic relatedness because it would enhance the weight of non-independent bands compared to the other bands.

9.3.4 Microsatellites Microsatellites or simple sequence repeat (SSR) loci, or the variable number of tandem repeats (VNTRs), or Sequence Tagged Microsatellites (STMS) or simple sequence length polymorphisms (SSLPs), are found throughout the nuclear genomes of most eukaryotes and to a lesser extent in prokaryotes (Varshney et al., 2005). Microsatellites range from one to six nucleotides in length (Van Oppen et al., 2000) and are classified as mono-, di-, tri-, tetra-, penta- and hexanucleotide repeats. Selkoe and Toonen (2006) stated that the sequences of di-, tri- and tetranucleotide repeats are the most common choices for molecular genetic studies. Goodfellow (1992) reported that microsatellites are repeated 5e20 times in the genome with a minimum repeat length of 12 bp. Microsatellites are highly polymorphic and abundant, often found in noncoding regions of genes. The mutation rate of microsatellites is high and has large numbers of alleles that vary in size at a single locus. DNA slippage polymerase and mismatch repair during replication resulted in diversity in microsatellite length. Variation in microsatellite length is detected by PCR using unique flanking primer sequences. Moore et al. (1991) and Rubinsztein et al. (1995) reported that PCR primers designed for one species can sometimes function in related species. Microsatellite markers are used for mapping genes controlling economic traits. Once a simple repeat region is identified, specific primers can be designed for PCR for genotyping by sequencing its immediate flanking regions. Polyacrylamide gel electrophoresis is done for determining the size of a microsatellite PCR product. Labeling of one of the two primers used in PCR is done with a radioactive or fluorescent tag. Codominance of alleles, high genomic abundance in eukaryotes, and random distribution throughout the genome are the major advantages of microsatellites. Since the technique is PCR-based, only less amount of DNA template is needed. Because of the use of long PCR primers, the reproducibility of microsatellites is high, and analyses do not require highquality DNA. Although microsatellite analysis is, in principle, a single-locus technique, multiple microsatellites may be multiplexed during PCR or gel electrophoresis if the size ranges of the alleles of different loci do not overlap; thus decreasing the analytical cost significantly.

9.3.4.1 Applications of microsatellites Microsatellites are the most popular genetic marker with several applications in population genetics, conservation biology, and evolutionary biology. The high level of polymorphism shown by microsatellites made them very informative markers. Microsatellites are widely used in population genetics and conservation biology, gene tagging and QTL analysis, hybridization and breeding, functional genomics, forensic science, disease diagnosis, taxonomic and phylogenetic studies, genome mapping, and sex determination (Abdul-Muneer, 2014).

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Unavailability of adequate primer sequences for the species of interest makes them difficult to apply in unstudied groups. High development costs are involved Mutations in the primer annealing sites lead to the occurrence of null alleles (no amplification of the intended PCR product), resulting in genotype scoring errors. Null alleles may result in a biased estimate of the allelic and genotypic frequencies and an underestimation of heterozygosity. Different forward and backward mutations lead to homoplasy at microsatellite loci, which may result in underestimation of genetic divergence. DNA slippage during PCR amplification results in the appearance of stutter bands (artifacts in the technique). These can complicate the interpretation of the band profiles due to the difficulty in size determination of the fragments, and heterozygotes may be confused with homozygotes. However, the interpretation may be clarified by including appropriate reference genotypes of known band sizes in the experiment.

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9.3.5 Minisatellites A minisatellite is a tract of repetitive DNA in which certain DNA motifs (ranging in length from 10 to 60 base pairs) are typically repeated 5e50 times. Minisatellites are notable for their high mutation rate and high diversity in the population, and they occur at more than 1000 locations in the human genome. Minisatellites are small sequences of DNA that do not encode proteins but appear throughout the genome hundreds of times, with many repeated copies lying next to each other. Minisatellites are prominent in the centromeres and telomeres of chromosomes, the latter protecting the chromosomes from damage.

9.3.6 Single nucleotide polymorphisms (SNPs) Genomic selection using the SNP markers is a powerful tool for the genetic selection of dairy animals (Seidel, 2009). Single nucleotide polymorphisms frequently called as SNPs are DNA sequence variations that happen when a single nucleotide: adenine (A), thymine (T), cytosine (C) or guanine (G) in the genome sequence is altered. SNP is a single basepair mutation at a specific locus, generally consisting of two alleles (where the rare allele frequency is >1%). The use of SNPs as markers for genetic analysis is increasing in comparison to other types of DNA markers because they are prevalent and provide more potential markers near or in any locus of interest; they are stably inherited making them more suited as long-term selection markers; SNPs located in coding regions directly affect the protein function thereby causing variation in important traits, and they are more suitable for high throughput genetic analysis, using DNA microarray technology (Lipshutz et al., 1999; Beuzen et al., 2000). Landegren et al. (1998) reported that SNP appears approximately at every 1000 bases in human genomic DNA. Frohlich et al. (2004) suggested that SNPs are the best genetic variation resource for population studies and genome mapping as they include more than 90% of all differences between the individuals. Genomic selection using the SNP markers is a powerful new tool for genetic selection (Seidel, 2009). The key challenge is to identify useful SNPs that can predict the breeding value of an animal.

9.3.6.1 Methods of SNP SNPs genotyping is performed using two main methods, the traditional and high throughput methods. The traditional gelbased approach utilizes standard molecular techniques like Amplification Refractory Mutation System (ARMS), Restriction Fragment Length Polymorphism (RFLP), Denaturing Gradient Gel Electrophoresis (DGGE), and Single-Strand Conformation Polymorphism (SSCP). High throughput methods include allele discrimination methods (Allele-Specific Hybridization, Allele-Specific Single-Base Primer Extension), High-throughput assay chemistry (Flap endonuclease discrimination, Oligonucleotide ligation), DNA arrays, pyrosequencing, and light cycler. Landegren et al. (1998) opined that the application of DNA microarray or ‘DNA-on-Chip’ technology to detect SNPs is a potentially powerful tool for high throughput DNA screening. Lipshutz et al. (1999) stated that up to 40,000 sequences could be screened at one time on a single slide. An experiment conducted by Wang et al. (1998) suggested that 2.3 Mb of genomic DNA were screened successfully to identify up to 3241 candidate SNPs, using more than 100 tiling microarrays. A tiling microarray consists of overlapping sets of four 25-mer-oligonucleotide probes derived from cDNA sequences, varying by one nucleotide in the central position.

9.3.6.2 Applications of SNP Traditional SNPs genotyping technologies are suitable for the study of few SNPs in the genome, which help to determine association between the SNPs and economic traits or disease. High throughput SNPs genotyping methods that provide a high density of SNPs are used in genomic selection, MAS, and QTL mapping. Genomic selection helps to calculate an accurate estimated breeding value (EBV) of animals before reaching sexual maturity, thereby identification of superior animals at an earlier age (Schefers and Weigel, 2012). SNPs are also used in Haplotype mapping, where sets of alleles or DNA sequences can be clustered so that a single SNP can identify many linked SNPs. A tag SNP is a representative singlenucleotide polymorphism in a region of the genome with high linkage disequilibrium. Tag SNPs are useful in wholegenome SNP association studies, in which hundreds of thousands of SNPs across the entire genome are genotyped. SNP identification exploits novel genomic selection methodologies in farm animals. Genomic selection results in reduced generation interval and increased selection intensity, thus designing a cost-effective breeding program.

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9.3.7 Allozyme markers Allozymes are allelic variants of enzymes encoded by structural genes and can be visualized through protein electrophoresis. Enzymes are proteins that consist of amino acids, some of which are electrically charged, and thus, have a net electric charge, depending on the stretch of amino acids comprising the protein. Zymograms are the banding pattern of isozymes, which can be readily interpreted in terms of loci and alleles, or they may require segregation analysis of progeny of known parental crosses for interpretation. Allozymes are codominant markers and are considered as molecular markers since they represent enzyme variants, and enzymes are molecules. However, allozymes are, in fact, phenotypic markers, and as such, they may be affected by environmental conditions. The banding profile obtained for a particular allozyme marker may change based on the type of tissue used for the analysis because a gene that is being expressed in one tissue might not be expressed in other tissues. Although molecular markers are based on differences in the DNA sequence, they are not environmentally influenced, which means that the same banding profiles can be expected at all times for the same genotype.

9.3.7.1 Applications Avise (1994) reported that this technique was developed for quantifying the genetic and geographic variation in wildlife populations, and it remains a cost-effective and straightforward method. Genetic variations caused by mutations are expressed as amino acid replacements due to changes in protein compositions and are resolved as bands (alleles) on electrophoretic gels (DeYoung and Honeycutt, 2005). They usually exhibit simple Mendelian inheritance and codominant expression, making genetic interpretations easy. Allozyme markers have high reproducibility. Allozyme markers are used in population genetics studies, including measurements of outcrossing rates, population divergence, and (sub) population structure. Allozymes are useful in the study of conspecific populations (animals belonging to the same species) and closely related species, and are, therefore, used for the determination of diversity in individuals and their relatives. They have been used, often in concert with other markers, to study the mode of genetic inheritance, to study interspecific relationships, and for fingerprinting purposes.

9.3.8 Mitochondrial DNA (mtDNA) Mitochondrial DNA is an extrachromosomal genome in the cell mitochondria, which resides outside the nucleus, and is inherited from the mother without paternal contribution (Emadi et al., 2010). Adams (1983) reported that mtDNAs have higher evolutionary rates in relation to the nuclear genome. The first significant part of the human genome to be sequenced was human mitochondrial DNA (Anderson et al., 1981). This sequencing revealed that the human mtDNA has 16,569 base pairs and encodes 13 proteins. Delsuc et al. (2003) and Hassanin et al. (2013) reported that mtDNAs are the backbone of phylogenetics and evolutionary biology since animal mtDNA evolves faster than nuclear genetic markers. It also helps to determine the relatedness of populations, and hence, has become important in anthropology and biogeography. Animal mtDNA is normally a circular, compact molecule about 17 Kb with little variation in size, containing 13 protein-coding genes, two rRNA, and 22 tRNA genes. This pattern is conserved among bilaterians, with few exceptions. Lavrov and Pett (2016) stated that there is a high variation in size, shape, gene content, and genetic code of mtDNA in nonbilaterians. Cameron et al. (2011) reported that the mtDNA of some animals is not a single circular molecule, but occurs as two or more circular or linear “chromosomes.” The number of these chromosomes can vary from two as in Liposcelis bostrychophila (Wei et al., 2012) to as high as 20 in the human body louse, Pediculus humanus (Shao et al., 2009). Similarly, an examination of the NCBI organelle genome database revealed that the GC content of animal mtDNA varies from 11% to 57%. Schutz et al. (1994) reported that the influence of maternal lines, maybe a sign of mitochondrial DNA differences, and probably be important for milk yield and successful reproductive performance in dairy cows (Bos taurus). Follow-up variations of mtDNA were studied in 36 maternal lines using an animal model for identifying the effect on milk yield and reproductive characteristics. It was assumed that cows within the maternal lineages, which were fixed by the herd book lineage, were equal in terms of the seeded nucleotide sequences. Thus it is revealed that polymorphism of mtDNA is associated with milk yield, reproduction, and health costs.

9.3.8.1 Functions and uses of mtDNA The most basic function of mitochondria is OXPHOS and is hence, known as the powerhouses of the cell. Its most important role is coding and synthesis of proteins, which are integral parts of enzymatic complexes that catalyze OXPHOS

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and is the function of mtDNA in all eukaryotes. However, mitochondria participate in various other, important cellular processes, such as apoptosis (Sinha et al., 2013), aging (Bratic and Larsson, 2013), signaling (Chandel, 2015), metabolic homeostasis, and biosynthesis of lipids and heme (Cheng and Ristow, 2013; Ahn and Metallo, 2015). Breton et al. (2014) reported that beyond the participation in OXPHOS, mtDNA-coded proteins exhibit a multiplicity of functions, which includes NAD2 that interacts with Src, a tyrosine kinase, which is critical for controlling important cellular functions (Gingrich et al., 2004). The males of several freshwater mussels carry a COX2 gene with a long extension of approximately 185 amino acid residues, which may help in the biparental transmission of mtDNA, known as doubly uniparental inheritance, DUI mechanism (Chakrabarti et al., 2007; Breton et al., 2014). However, there are mtDNA-encoded peptides that do not participate in OXPHOS but might play important roles in other cellular functions. Avise (2012) reported that animal mtDNA possesses characteristics that make it an ideal genetic marker. Maternal inheritance and absence of recombination are very desirable properties for the reconstruction of phylogenetic histories since it enables tracing each lineage as a single evolutionary history. The existence of conserved and less conserved regions within the same molecule and the elevated mutation rate relative to the nuclear DNA makes mtDNA suitable for comparison among individuals from the same population, as well as among distantly related species. These intrinsic properties of mtDNA are powerful aid for population genetic studies. The alternation of variable and conserved regions on the same molecule allows for the design of universal primers, which is able to amplify fragments of the mtDNA of any species without prior knowledge about the species mtDNA. Multiple copies of mtDNA within each cell made the amplification of the mtDNA easier than parts of the nuclear DNA. Direct sequencing of the PCR product was made feasible by the homoplasmy of mtDNA, unlike nuclear genes, where the maternal and paternal alleles need to be separated before sequencing. Both the intrinsic properties and the technical ease made mtDNA the most popular genetic marker before the advent of large-scale sequencing.

9.3.8.2 Maternal transmission mtDNA is not always maternally transmitted. Zouros (2013) and Breton et al. (2007) reported doubly uniparental inheritance (DUI), a case of paternal transmission in several species of molluscan bivalves. In this method of inheritance, females will transmit their mtDNA to both male and female offspring, and males will transmit their mtDNA only to male offspring resulting in co-occurrence in the same species of two independently evolving mtDNA lineages, one that is transmitted through the eggs and another through the sperm. As a result, females are homoplasmic for the maternal mtDNA but might contain low amounts of paternal mtDNA, and produce eggs with only the maternal mtDNA. However, males are heteroplasmic for both the maternal and the paternal mtDNA but produce sperm that contains only the paternal mtDNA. Sato and Sato (2013) stated that the mechanisms which confirm the maternal transmission of mtDNA differ in each organism. In mammals, sperm mitochondria are ubiquitinated and subsequently destroyed (Sutovsky et al., 1999); in Drosophila, mitochondria are destroyed during spermatid formation (DeLuca and O’Farrell, 2012); in Oryzias latipes, the mitochondria of the sperm are actively destroyed (Nishimura et al., 2006), and in Caenorhabditis elegans the sperm’s mitochondria are destroyed through autophagy (Sato and Sato, 2011; Al Rawi et al., 2011). Maternal inheritance of mtDNA results in homoplasmic individuals, i.e., individuals having a single type of mtDNA. Mishra and Chan (2014) opined that homoplasmy is reinforced by pre- and postfertilization bottlenecks. The prefertilization bottleneck occurs during oogenesis, where the number of mitochondria is critically reduced in the germline, before the maturation of the oocyte. The postfertilization bottleneck occurs between the zygote formation and the blastocyst embryonic stages, during which there is intense cell division but suppression of mitochondrial proliferation, a mechanism that leads to a reduced number of mitochondria per cell.

9.3.8.3 Heteroplasmy Heteroplasmy may result through different not mutually exclusive routes. First, the egg may be heteroplasmic, consisting of two or more different types; this is the case of mother-inherited heteroplasmy. Second, somatic mutations may happen during mtDNA replication in somatic cells. Given the tremendous amount of mtDNA copies per individual (a diploid organism may contain billions of cells, and each cell consists of two copies of nuclear genes but hundreds or thousands of copies of mtDNA) and the elevated mutation rate of mtDNA in animals, each individual contains unavoidably numerous mutated forms of mtDNA which it has inherited from its mother. Finally, leakage of paternal mtDNA can be a significant source of heteroplasmy. The mitochondria of the sperm may escape destruction in the egg during fertilization so that the embryo will be heteroplasmic for a maternal and a paternal mtDNA molecule. Kondo et al. (1990) and Dokianakis and Ladoukakis (2014) suggested that heteroplasmy will be more common in progeny from heterospecific than from homospecific crosses.

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The most frequently used technique for the detection of heteroplasmy includes PCR-amplification of a specific segment of mtDNA molecule and further sequencing, either directly or after cloning. If the targeted mtDNA pool contains a predominant molecule and some other types in low frequencies, the traditional Sanger sequencing will not reveal the presence of the rare molecules. Only specific primers designed for the rare molecules could detect them when using direct sequencing. A large number of clones from the PCR product need to be sequenced, or next-generation sequencing (NGS) should be applied for identifying the presence of rare molecules.

9.3.8.4 Recombination Recombination in plants and fungi mtDNA has been reported in the early 80s of the past century (Stern and Palmer, 1984; Taylor, 1986). Wilson et al. (1985) reported that animal mtDNA was considered as a nonrecombining genome for decades. But experiments conducted by Thyagarajan et al. (1996) revealed that animal mitochondria contain the enzymatic apparatus for recombination. Ladoukakis and Zouros (2001) reported the first direct evidence for recombination in mussels. Kraytsberg et al. (2004) reported mtDNA recombination in humans. Ma and O’Farrell (2015) reported mtDNA recombination in Drosophila. This was done by either direct sequencing or utilizing data deposited in Gen Bank (Piganeau et al., 2004; Tsaousis et al., 2005).

9.3.8.5 Applications of mtDNA markers Mitochondrial DNA (mtDNA) marker is preferred in constructing phylogenies and inferring evolutionary history, and is, therefore, ideal for within- and between-species comparisons (Emadi et al., 2010). mtDNA polymorphisms have also been used in genetic diversity analyses. Nijman et al. (2003) reported that mtDNA markers provide a rapid way of detecting hybridization between livestock species or subspecies.

9.3.9 DNA barcoding markers A DNA barcode is a short DNA sequence from a standardized region of the genome used for identifying species. Identification of species by DNA barcoding depends upon a suitable DNA barcode, which refers to a standardized sequence (usually less than 1000 bp) of the genome. The barcode must have universality so that it can be easily amplified from diverse species, and it must contain few insertions or deletions to ease the sequence alignment. Its mutation rate should be sufficient enough to generate a barcoding gap, which means that the maximum intraspecific variation is less than the minimum interspecific distance. Hebert et al. (2003) reported that the aim of DNA barcoding is the large-scale screening of one or more reference genes to assign unknown individuals to species and to enhance the discovery of new species. Tautz et al. (2002) conducted the first application of using the DNA sequences in systematic biological taxonomy. Hebert et al. (2003) suggested the use of DNA barcoding in a single mtDNA gene, mitochondrial cytochrome c oxidase I (COI), as a common sequence in animal DNA barcoding studies. Goodfellow (1992) reported that DNA barcoding has a high accuracy of 97.9%. Morin et al. (2004) opined that DNA barcoding provides a quick and convenient identification strategy for animal genetic diversity studies. Some organisms cannot be identified with COI due to low evolution rates of COI sequences in some species. Hajibabaei et al. (2006) reported that COI is an mtDNA sequence of maternal origin, which could bias species diversity. COI is highly conserved across species employing oxidative phosphorylation for metabolism. Several studies have revealed that COI-based DNA barcoding can delimit diverse animal species, indicating the high rates of sequence change at the species level and constraints on intraspecific divergence in COI sequence. A full-length barcode (658-bp) region of the COI gene is suggested for the analysis of fresh and well-preserved animal tissues due to the feasibility of PCR amplification.

9.3.9.1 Animal identification by DNA barcoding Two basic steps are involved in the process of DNA barcoding. First, a barcode library of identified/reference species is built; second, barcode sequence of the unknown sample is compared with the barcode library for its identification. After the extraction of DNA from the tissue samples, appropriate DNA barcodes must be selected and then amplified through polymerase chain reaction (PCR). The amplified regions can be sequenced by conventional methods or NGS according to the sample complexity and then compared with existing sequences from the reference database. DNA barcode is generated in the form of a chromatogram. The chromatogram is a visual representation of the DNA sequence produced by the sequencer. This barcode can be stored in the database for future use or can be used for comparative analysis to detect the intraspecific and interspecific sequence divergences (Kress and Erickson, 2012).

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9.4 Marker assisted selection (MAS) Selection is one of the most important tools which help to improve the performance of animals. The addition of genomic information to phenotypic information for enhancing the response to selection is called Marker Assisted Selection (MAS). Sax (1923) reported the concept of MAS as an aid to selection using the information from polymorphic loci. Wells et al. (1998) stated that MAS is a method where the presence of desirable genes is indicated by marker genes. Ribaut and Ragot (2006) opined that MAS is an indirect selection process in which the trait of interest is selected not only based on the trait itself but also on the marker linked to it. MAS can be based on DNA in linkage equilibrium with a quantitative trait loci (QTL) which is known as LE-MAS, or molecular markers in linkage disequilibrium with a QTL which is known as LDMAS, or based on the selection of the actual mutation causing the QTL effect which is known as Gene assisted selection (GAS) (Dekkers, 2004). Linkage Equilibrium (LE) indicates genotype frequencies at one locus are independent of genotype frequencies at the second locus. That is when the haplotype frequencies are equal to the product of their corresponding allele frequencies; it means the loci are in linkage equilibrium. Linkage disequilibrium (LD) indicates the nonrandom association of alleles between two loci. We can deduce linkage disequilibrium for each haplotype as the deviation of observed haplotype frequency from its corresponding allelic frequencies expected under equilibrium. Direct markers are preferred for effective implementation of MAS, followed by LD and LE markers, the latter requiring within-family analysis and selection. Ease of application and potential for extra genetic gain is greatest for direct markers, followed by LD markers, but is antagonistic to ease of detection, which is greatest for LE markers. Thus, the ease and ability to use markers in selection is opposite to their ease of detection and increases from direct markers to LD markers and LE markers. All three types of MAS are being used in the livestock industries (Dekkers, 2004). MAS is applied for the indirect selection of superior breeding animals and depends on identifying the association between a genetic marker and linked Quantitative trait loci (QTL). Genetic evaluation and selection can be improved by combining genetic information at markers and QTL with the phenotypic information. MAS helps in the direct measurement of the effect of genes on production from the genetic makeup of the animal rather than from the phenotype. Response to selection was benefited by the integration of the traditional or conventional selection methods with molecular genetics methods. MAS facilitates multiple estimated QTL effects and multiple trait selection to make better decisions for animal improvement. Information from marker’s genotypes combined with information on animal’s phenotype aids in MAS. MAS depends on identifying the association between the marker and linked QTL. The association between the genetic marker and linked QTL depends on the distance between the marker and target traits. Once the marker linked to QTL is identified, they can be used in the selection program. This use of marker in the selection program is called MAS, which is the most widely used application of marker systems in breeding. MAS aims at increasing the genetic gain by enhancing the accuracy of selection. Most of the animal breeding schemes rely on the high accuracy of selection (progeny testing with 90% accuracy of selection). MAS is useful for traits, where the accuracy of conventional selection is very low, like traits with low heritability, traits with few recording (due to expensive recoding), traits which are measurable late in life (milk production in fifth lactation or life time milk production in dairy cattle and buffaloes), traits which are not available at the time of selection (slaughter quality traits), and traits that require expensive and risky challenge testing (disease resistant traits).

9.4.1 Applications of MAS The application of MAS in breeding programs depends on the availability of variable marker information, different effects on multiple traits, and genotypic information that helps in improving commercial breeding activities. MAS is an aid in the detection of genes for disease resistance, product quality (Ashwell et al., 1997), and genetic disorders (Georges et al., 1993). Neeteson et al. (1999) reported that MAS helps in improving stress resistance, feather pecking, longevity, and desired behavioral characteristics. Application of MAS in disease resistance: MAS allows selection for disease resistance and enables highly accurate selection, which is unaffected by environmental factors. Large numbers of samples need to be screened for the genes conferring resistance to a given disease. Distinction between lines, which are resistant and susceptible to that particular disease, is possible. Application of MAS in selection and breeding: Selection with the help of genetic markers information is known as MAS. Dekkers (2004) stated that MAS can be used either by linking MAS disequilibrium or through gene assisted selection in the livestock breeding industry. Liu and Cordes (2004) opined that genomic research and QTL mapping result in MAS for precise and efficient selection. MAS enables selection without the expense of progeny testing. Selection of related

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individuals that do not express the traits like milk production and egg-laying in males is possible through MAS that can also be used in introgression strategies for the selection of traits to be introgressed, as well as against undesirable traits (Hillel et al., 1990). MAS is useful for highly heritable traits of large effect, and such traits are already fixed with nearoptimal alleles in commercial lines. Ruane and Colleau (1996), Spelman and Garrick (1997) reported the economic advantage of MAS in dairy cattle. MAS is applied with Multiple Ovulation Embryo Transfer (MOET) technology and progeny testing. An animal’s true genotype can be estimated with the help of knowledge of genes located at QTL. The information available at QTL adds to the accuracy of breeding value estimation. Genes with larger genetic effects at QTL can be used more specifically in breeding programs. MAS can increase the rate of genetic response from 5% to 64% in animal breeding populations based on the trait being selected and information on a marker and QTL (Hayes and Goddard, 2003). Ruane and Colleau (1996) found 6%e15% increase in response to selection for milk production in cattle by using MOET in the first six generations of selection. Molecular markers are used to identify loci or chromosomal regions that affect single gene traits and also QTLs. Combining phenotypes with genetic markers is a promising approach, which helps in the improvement of health and welfare traits in farm animals. MAS is currently being used in commercial livestock breeding. Large-scale genotyping methods and infrastructure, which enable the generation of hundreds of thousands of molecular data at a reasonable cost, will be necessary for making MAS effective in a large breeding population. MAS helps in increasing genetic gain as compared to traditional breeding programs and reduces the cost of progeny testing by an early selection of the potential young bulls. MAS eases the exploitation of existing genetic diversity in breeding populations and helps in improving desirable traits in livestock.

9.5 Conclusion Improvement of livestock depends on the selective breeding of the individual animals with superior phenotype. Molecular markers are a potential aid to animal breeding, which helps in identifying the genetic makeup of an animal and thereby predicting its performance. Molecular markers allow easy selection of a number of valuable traits more directly. They have played a significant role in animal breeding by maximizing selection, particularly for a low heritable or expensive trait or traits, which are difficult to measure phenotypically or traits that are expressed later in life. Application of molecular markers includes conservation of genetic diversity, identification of disease carriers, parentage determination, MAS, transgenesis, sex-determination, creation of genetic linkage map, and many more. The most widely used application of marker systems in breeding is the MAS scheme that can be designed to shorten the generation interval considerably and still maintain a high accuracy of selection through the use of marker information. Utilization of marker-based information for genetic improvement of animal depends on the choice of an appropriate marker. In future, molecular markers may serve as a potential tool for the evaluation and manipulation of existing germplasm in order to select and create the desired trait in animals, which, in turn, results in genetic improvement of animals.

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Further reading Ajmone-Marsan, P., Vecchiotti-Antaldi, G., Bertoni, G., Valentini, A., Cassandro, M., Kuiper, M., 1997. AFLPÔ markers for DNA fingerprinting in cattle. Anim. Genet. 28 (6), 418e426. Al-Samarai, F.R., Al-Kazaz, A.A., 2015. Applications of molecular markers in animal breeding: (A). Am. J. Appl. Sci. Res. 1 (1), 1e5. Bardakci, F., 2001. Random amplified polymorphic DNA (RAPD) markers. Turk. J. Biol. 25 (2), 185e196. Braun, A., Little, D.P., Reuter, D., Muller-Mysok, B., Koster, H., 1997. Improved analysis of microsatellites using mass spectrometry. Genomics 46 (1), 18e23. Breton, S., Ghiselli, F., Passamonti, M., Milani, L., Stewart, D.T., Hoeh, W.R., 2011. Evidence for a fourteenth mtDNA-encoded protein in the femaletransmitted mtDNA of marine mussels (Bivalvia: mytilidae). PLoS One 6 (4), e19365. Caetano-Anolles, G., Brant, B., 1991. DNA amplification fingerprinting using very short arbitrary oligonucleotide primers. Biotechnology 9 (6), 553. Christie, J.R., Schaerf, T.M., Beekman, M., 2015. Selection against heteroplasmy explains the evolution of uniparental inheritance of mitochondria. PLoS Genet. 11 (4), e1005112. Ewing, B., Green, P., 2000. Analysis of expressed sequence tags indicates 35,000 human genes. Nat. Genet. 25 (2), 232. Gholizadeh, M., Mianji, G.R., Zadeh, H.S., 2008. Potential use of molecular markers in the genetic improvement of livestock. Asian J. Anim. Vet. Adv. 3 (3), 120e128. Hadrys, H., Balick, M., Schierwater, B., 1992. Applications of random amplified polymorphic DNA (RAPD) in molecular ecology. Mol. Ecol. 1 (1), 55e63. Haff, L.A., Smirnov, I.P., 1997. Multiplex genotyping of PCR products with MassTag-labeled primers. Nucleic Acids Res. 25 (18), 3749e3750. Hoeschele, I., Meinert, T.R., 1990. Association of genetic defects with yield and type traits: the weaver locus effect on yield. J. Dairy Sci. 73 (9), 2503e2515. Ladoukakis, E.D., Zouros, E., 2017. Evolution and inheritance of animal mitochondrial DNA: rules and exceptions. J. Biol. Res. Thessalon 24 (1), 2. Lander, E.S., Botstein, D., 1989. Mapping mendelian factors underlying quantitative traits using RFLP linkage maps. Genetics 121 (1), 185e199. Lightowlers, R.N., Chinnery, P.F., Turnbull, D.M., Howell, N., 1997. Mammalian mitochondrial genetics: heredity, heteroplasmy and disease. Trends Genet. 13 (11), 450e455. Ma, H., Xu, H., O’Farrell, P.H., 2014. Transmission of mitochondrial mutations and action of purifying selection in Drosophila melanogaster. Nat. Genet. 46 (4), 393. Montaldo, H.H., Meza-Herrera, C.A., 1998. Use of molecular markers and major genes in the genetic improvement of livestock. Electron. J. Biotechnol. 1 (2), 15e16. Neuner, S., Emmerling, R., Thaller, G., Gotz, K.U., 2008. Strategies for estimating genetic parameters in marker-assisted best linear unbiased predictor models in dairy cattle. J. Dairy Sci. 91 (11), 4344e4354. Nicholas, F.W., 1996. Genetic improvement through reproductive technology. Anim. Reprod. Sci. 42 (1e4), 205e214. Pirchner, F., 1983. Population Genetics in Animal Breeding (San Francisco). Rao, K.A., Bhat, K.V., Totey, S.M., 1996. Detection of species-specific genetic markers in farm animals through random amplified polymorphic DNA (RAPD). Genet. Anal. Biomol. Eng. 13 (5), 135e138. Rohrer, G.A., Alexander, L.J., Keele, J.W., Smith, T.P., Beattie, C.W., 1994. A microsatellite linkage map of the porcine genome. Genetics 136 (1), 231e245. Rokas, A., Ladoukakis, E., Zouros, E., 2003. Animal mitochondrial DNA recombination revisited. Trends Ecol. Evol. 18 (8), 411e417. Sharpley, M.S., Marciniak, C., Eckel-Mahan, K., McManus, M., Crimi, M., Waymire, K., Chalkia, D., 2012. Heteroplasmy of mouse mtDNA is genetically unstable and results in altered behavior and cognition. Cell 151 (2), 333e343. Stewart, J.B., Chinnery, P.F., 2015. The dynamics of mitochondrial DNA heteroplasmy: implications for human health and disease. Nat. Rev. Genet. 16 (9), 530e542.

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Teneva, A., 2009. Molecular markers in animal genome analysis. Biotechnol. Anim. Husb. 25, 1267e1284, 5e6e2. Vignal, A., Milan, D., SanCristobal, M., Eggen, A., 2002. A review on SNP and other types of molecular markers and their use in animal genetics. Genet. Sel. Evol. 34 (3), 275. Vos, P., Hogers, R., Bleeker, M., Reijans, M., van de Lee, T., Hornes, M., Zabeau, M., 1995. A new technique for DNA fingerprinting. Nucleic Acids Res. 23 (21), 4407e4414. Wakchaure, R., Ganguly, S., Praveen, P.K., Kumar, A., Sharma, S., Mahajan, T., 2015. Marker assisted selection (MAS) in animal breeding: a review. J. Drug Metabol. Toxicol. 6, e127. Williams, J.L., 2005. The use of marker-assisted selection in animal breeding and biotechnology. Rev. Sci. Tech. Off. Int. Epizoot. Paris 24 (1), 379.

Chapter 10

Genomic selection: a molecular tool for genetic improvement in livestock D.N. Das, T. Karuthadurai and Shanmugapriya Gnanasekaran Genetics Laboratory, Dairy Production Section, ICAR-National Dairy Research Institute (SRS), Southern Regional Station, Bangalore, Karnataka, India

10.1 Introduction This chapter presents issues pertaining to the genetic improvement of livestock, especially using molecular tools. It covers aspects from basic population to quantitative genetics to molecular genetics and genomics, and their application in animal breeding. Genetic improvement takes place as an outcome of raising the frequency of desirable genes and declining the frequency of the undesirable genes. Genetic improvement through selection can occur in spite of the type of management, feed, or facilities used in a herd. However, the greatest genetic improvement is most likely to be realizing when animals are reared in a herd environment similar to the commercial production environment. Genetic improvement does not result from changing the management, facilities, feeds, etc., although, by making such changes, the herd average for a particular trait may improve. The reason is that changing these environmental factors does not change either the genes carried by the animals or the genotypes obtained from these genes; therefore, genetic improvement will not occur. The young male animals are selected based on the progenies’ performances. It is obvious that the direct recording of data based on observations of the average milk yield of a herd provides very little indication about the genetic progress of the herd. There are many reasons as to why the yield of a herd increases over a period of time either by improving herd management skill or through providing higher input in terms of feed. In the first instance, it seems to be possible to measure the effectiveness of such exogenous changes or modifications on milk yield by considering the yield of the same cow in successive years. After subtracting the environmental effects, genetic changes are determined. In this method, further, it is mentioned that two factors viz., age of the animal and management changes are to be taken into consideration for estimating the precise genetic improvement due to genetic cause. Suitable correction factors for different environments or management systems and various age groups are needed. Correction factors may be taken from estimates calculated in other herds. The effect of age is not independent of management. For example, a cow matured at an early age by heavy feeding will not show proper age effect in comparison to cows managed with a lower plane of nutrition. Hence, the correction factors must be fitted to the management of the herd. In a herd, the animals which are maturing quickly either due to genetic or managemental in comparison to another group of animals, suitable correction factors for nongenetic effects are to be considered. The concept of animal breeding toward true genetic improvement is based on the basic concept of heritability, which relates the phenotype of an animal to genotypic value, or in narrow sense, regression of genotypic value on phenotype.

10.2 Conventional selection Although traditional breeding methods have been effectively used for selection of superior animals for many economic importance traits, with these methods, the accuracy of breeding value always remains doubtful due to low heritability and without having any prior knowledge about the molecular level. For obtaining genetic improvement, the breeding value of the animals must be estimated from the measurable or quantifiable phenotypes of the animals themselves (or) those of their relatives. The phenotypes may be performance records on the prospective replacement stock, performance records on the relatives of the prospective replacement stock, or a visual evaluation of the animals or their relatives. Visual evaluation is

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not recommended for traits that can be measured. In any case, there must be a mechanism in place that will permit the ranking of the tested animals from best to worst within a contemporary group for the traits under selection. A contemporary group consists of animals of the same breed and sex that were furrowed within three or four weeks of each other, housed together, fed the same feed, and managed as much alike as possible. The recommended method for comparing the genetic merit of prospective breeding animals from a contemporary group is to estimate their breeding values (EBV) or their expected progeny differences (EPD) from their own performance records and also the performance records of their relatives. The animals with the best-estimated breeding values should be retained as replacement animals, and those with the poorest estimated breeding values should be culled from the herd. Marker Association with QTLs and genome-based selection are the latest edition in modern breeding practices in farm animals.

10.3 Selection - the major tool for genetic improvement The ultimate goal of a breeding program is the genetic improvement of traits defined in the breeding objective for the animal population. The major tool to achieve this is to select the best animals as parents to produce the next generation, and among those parents, also decide the ones that should have the largest number of offspring. With a successful selection, the progeny generation will, on average, be better than the average of the population from which the parents were chosen and genetic progress is obtained. The gains are accumulated when we continue to select the best animals in each generation. A continuous, long-lasting genetic improvement in traits included in the breeding goal is thus achieved. Genetic change can be attained depending on a number of factors. First of all, the traits that are to be improved must show additive genetic variation, and we need to be able to identify the best animals. It also matters as to how many traits we include in the breeding goal, what proportion of animals are selected, how intensively they are used, how long the generation interval will be, etc. Moreover, we need to be aware of the potential existence of genotype-environment interactions, and that a specific genotype is not always the best one in all environments.

10.4 Principles of selection Selection is defined as an improvement in which certain individuals in a population are preferred to others for the production of the next generation. Selection is of two types viz., natural causes by various forces of nature, and artificial, or happenings due to human intervention. Although no new genes are created by the selection, under selection pressure, there is a tendency for reduction of frequency of the undesirable genes; thereby, the frequency of the more desirable genes is enhanced. Thus the main genetic effect of selection is to change gene frequencies, although there may be a chance for an increase in homozygosity of the desirable genes in the population as progress is made in selection.

10.5 Natural selection In nature, the main force responsible for selection is the survival of the fittest in a particular environment. Natural selection is of interest because of the parent’s effectiveness and the principle involved. Some of the most interesting cases of natural selection are those involving man himself. All races of humanity that now exit belong to the same species because they are interfertile or have been in all instances where matings have been made between them. All races of hummanity in existence had a common origin, and at one time, probably all humans had the same kind of skin pigmentation, which kind, we have no sure way of knowing. As the number of generations of humans increased, mutations occurred in the genes affecting pigmentation of the skin, causing genetic variations in this trait over a range from light to dark or black. Humans began to migrate into the various parts of the world and lived under a wide variety of climatic conditions of temperature and sunshine. Fitness determines the selection of character under natural selection. The fitness of an animal is the contribution of genes that transmit to the next generation. Relative fitness means the fitness of an animal relative to the population mean. If the population does not expand or contract in terms of number, then the mean fitness of animals is one and absolute fitness and relative fitness are the same. The fitness of an individual animal is the final outcome of all its developmental and physiological processes. The differences between individuals in these processes are seen in the variation of measurable attributes, which can be studied as metric characters. Fitness in mammals is divided into two major components, i.e., the total number of offsprings and the quality of these offsprings. The hierarchy of causes of variation on metric characters under fitness reveals the viability of offspring, litter size, number of litters and milk yield, and maternal behavior. It is the complicated progress, and many factors determine the proportion of individuals that will be reproduced; among these factors are differences in mortality of the individuals in the population, especially early in life, the difference in the duration of the period of sexual activity and the degree of sexual activity itself, and difference in genres of the fertility of individuals in the population.

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10.6 Artificial selection Artificial selection is that which is practiced by man. Thereby, man determines, to a great extent, the animals that will be used to produce the next generation of offsprings. Some researchers have divided selection in farm animals into two kinds, one known as automatic and the other as a deliberate selection. Litter size in swine may be used as an illustration of the meaning of these two terms. Here automatic selection would result from differences in litter size even if parents were chosen entirely at random from all individuals available at sexual maturity. Under these conditions, there would be twice as much chance of saving offspring for breeding purposes from a litter of eight than from a litter of four. The automatic selection here differs from natural selection only to the extent that the size of the litter in which an individual is reared influences the natural selective advantage of the individual for other traits. Deliberate selection, in this example, is the term applied to selection in swine for litter size above that which was automatic. In one study by Dickerson and coworkers, involving selection in swine, most of the selection for litter size at birth was automatic and very little deliberate; however, the opportunity for deliberate selection among pigs was utilized more fully for growth rate. Artificial Selection is a form of selection in which we actively choose the desirable traits that are passed on to the offsprings. Humans have used selective breeding long before Darwin’s Postulates and the discovery of genetics. Farmers chose cattle with beneficial traits such as larger size or producing more milk, and made them breed; and although they may have known nothing about genes, they knew that the beneficial traits could be hereditary. Farmers can breed animals in order to improve productivity, and thus, profits. For example, dairy farmers will look for the cows that can produce the most milk and only breed those cows. These cows then pass their genes that contribute to higher milk production onto their offspring, increasing productivity in each generation for the farmers. Selection based on many traits or multitraits selection in terms of progeny testing for male selection and selection indices for female selection becomes effective. A definite difference between breeds and types of farm animals within a species is proof that artificial selection has been effective in many instances. This is true, not only from the standpoint of color patterns that exist in the various breeds but also from the standpoint of differences in performances that involve certain quantitative traits. For instance, in dairy cattle, there are definite breed differences in the amount of milk produced and in butterfat percentages of the milk.

10.7 Selection for additive gene action Additive gene action plays a pivotal role in the expression of quantitative traits of great economic importance such as gain, milk production, carcass quality, and others. When this kind of gene action is important, selection merely becomes a matter of finding those individuals that are superior for the traits and then mating the best, year after year. If this is done, eventually, most of the contributing genes could be combined into one breed or one herd, although this depends upon efforts to make the selection as effective as possible.

10.8 Selection for multiple alleles It is not known for certain if one or more series of multiple alleles affect economic traits in farm animals, although they are known to affect traits such as coat color and blood type in cattle. If a series of multiple alleles such as A1, A2, and A3 did exist, it is possible that one might have more favorable effects on traits than the other two. The genetic improvement would come about by culling the less desirable animals for the particular trait and mating the best. By doing this, the frequency of the desirable allele should be increased in the population.

10.9 Selection for epistasis It is the interaction between genes, which are not alleles, and this interaction may be several kinds. The action may be either complementary or inhibitory, but we do not know for certain in what manner the genes may act, as far as their influence on the important economic traits in farm animals are concerned. If epistatic action is of a complementary nature, and it probably is in many cases, the advantage could be taken of this kind of gene action by forming inbred lines and then testing them in crosses to find those that are superior in this respect. The genetic improvement obtained in each generation of selection depends on: 1. Accuracy of the phenotypic evaluation(s) or the performance records in predicting the animal’s true breeding value or genotype (A)

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2 Intensity or degree of selection based on the phenotypic evaluations practiced on animals (I) 3 Amount of genetic or true breeding value variation among animals for the trait under selection (G) Genetic improvement/generation ¼ A  I  G. It is to be noted that if any one of these three factors is equal to zero, the product of the calculation is zero, indicating that no genetic progress can be expected regardless of the size of the other two factors. Heritability is the proportion of the additive genetic variation to the total variation. Heritability is important because, without genetic variation, there can be no genetic change in the population. Alternatively, if heritability is high, genetic change can be quite rapid. Using an increasing scale from 0 to 1, a heritability of 0.75 means that 75% of the total variance in a trait is controlled by additive gene action. With very high heritability, just the record of a single individual’s traits can be easily used to create an effective breeding program. Four main factors that affect the rate of genetic change in a herd are genetic variation, selection intensity, generation interval, and accuracy of selection. Genetic variation is the variability of the genetics in the herd. An inbred herd will have a low genetic variation, and therefore, will have a low rate of genetic change.

10.10 Selection intensity It refers to how many of the animals in the herd are culled. The more intense the culling program, the greater is the genetic gain. This occurs because there will be a greater percentage of exceptional animals selected to be used in the breeding program. Selection differential is the phenotypic average difference of the selected parent animals (Ps) from the population average (P0 ). Selection intensity (i) is the selection differential expressed in terms of phenotypic standard deviations (s); i.e., the ratio of (PsP0 ) over s. Selection is used in predicting genetic response due to selection because it can be related to that percent of the population saved as parents.

10.11 Generation interval The average age of an animal when replacement progeny is born refers to how long it takes to replace one generation with the next. This also depicts the average age of the parents at birth of their offspring that, in their turn, will produce the next generation of breeding animals. The generation interval facilitates to calculate the genetic response per year instead of per generation.

10.12 Selection accuracy It refers to how accurate we are when selecting the animals to keep as replacements for the current herd. There is a chance of a 10%e30% increase in accuracy of selection through gene-based and genome-based selection if other conditions remain optimum.

10.13 Phenotypic value The value observed when the character is measured on an animal is the phenotypic value of that animal. The partition of phenotypic value is into a component attributable to the influence of genotype, as well as the environment. The genotype is the specific assembly of genes possessed by the individual, and the environment depicts all nongenetic circumstances that influence the phenotypic value. Two components of value related to genotype and environment are the genotypic value and environmental deviation, respectively. P ¼ GþE where P is the phenotypic value, G is genotypic value, and E is environmental deviation. Considering a single locus with two alleles viz., A1 and A2, we consider the genotypic value of one homozygote as þa, of the other homozygote as e a, and that of the heterozygote as d. We, thus, have a scale of genotypic value, as mentioned below. The origin or point of zero value on this scale is midway between the values of two homozygotes. The value d of the heterozygote depends upon the degree of dominance. If there is no dominance, d ¼ 0; if A1 is dominant over A2, d is positive, and if A2 is dominant over A1, d is negative. If dominance is complete, d is equal to þa or ea, and if there is overdominance, d is greater than þa or less than ea. hence, the degree of dominance may be expressed as d/a.

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A2A2

A1A2

Genotypic value -a

0

145

A1A1

d

+a

10.14 Breeding value Progeny receives genes from both the parents, and hence, the average effect of genes determines the mean genetic value of the progeny. The value of an individual judged by the mean value of its progeny is called the breeding value of the animal. Thus for a single locus with two alleles, the breeding value of the genotype is as follows: Genotype

Breeding value

A1A1 A1A2 A2A2

2a1 ¼ 2qa a1 þ a2 ¼ (q e p) a 2a2 ¼ 2pa

10.15 Population mean The mean value in the whole population is obtained by multiplying the value of each genotype by its frequency and summing over the three genotypes. Genotypes A1A1 A1A2 A2A2 Sum

Frequency 2

p 2pq q2

Genotypic value

Frequency 3 genotypic value

þa d a

p2a 2pqd q2a a (pq) þ 2dpq

The population means is the sum of frequency  genotypic value. Thus, population means is as follows: M ¼ aðp  qÞ þ 2dpq

10.16 Average effect For understanding the properties of the population connected with pedigree toward transmission of value genetic material from parents to offspring, which cannot be done through genotypic value alone, as parents pass on their genes and not their genotypes to the next generation; hence, a new genotype is created afresh in each generation. Hence, a new measure of value is required, which will refer to the gene and not the genotype. Thus, we will be enabled to assign a breeding value to individual, a value associated with the gene carried out by the individual and transmitted to its offspring. The value associated with gene as distinct from genotype is known as the average effect.

10.17 Dominance deviation When a single locus only is under consideration, the difference between genetic value G and the breeding value A of a particular genotype is known as the dominance deviation D, so that G ¼ AþD If the genotype deals with more than one locus, the genotypic value may contain an additional deviation due to nonadditive combination, which is also called interaction or epistasis effect. Hence, for all loci, we can derive the equation as follows: G ¼ AþDþI

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10.18 Genetic control on production traits The functionality of milk production traits, lactation milk yield, and milk composition is governed by genes. The genetic value of a trait signals the probability that the genes responsible for that trait will be transferred to any offspring. Hence, dairy farmers/entrepreneurs selecting animals for the breeding stock would be typically more concerned with an animal’s true genetic merit than phenotypic value. Further, it becomes a challenge for breeders to retain the best animals having culled the inferior toward choosing high-quality milk production traits, as well as any other desirable attributes. Two main reasons for neglecting fitness traits of cows associated with increased genetic merit for milk yield highlights (a) fitness traits are given least importance in the construction of selection indices because of lower heritability or difficult to record and (b) use of inappropriate breeding program without considering appropriate selection measures and inbreeding depression (Goddard, 2009). The appropriate strategy for any breeding program would, therefore, be to set suitable selection goals, which suit the prevailing production system rather than ambitious performance objectives, which are difficult to achieve under the present environment. Based on different geoclimatic situations, an area-specific approach utilizing the available resources and taking into account the prevailing constraints appears to be the only reasonable and sustainable solution. Such an approach would also suggest in situ conservation and improvement of farm animal genetic resources, the only viable and practical genetic improvement method in less developed countries, especially with more number of breed/genetic differences than ex situ or cryopreservation approaches.

10.19 Genetic control on reproductive traits The decrease in the reproductive performance of lactating dairy cows partly indicates the failure of genetic selection for reproductive traits even though, fertility has dropped in lactating dairy cows, and even it remained better in dairy heifers. There is increasing evidence to suspect that embryo quality, corpus luteum (CL) function and endometrial environment are major factors affecting fertility status in lactating dairy cows (Richard Pursley et al., 1998). It is well known that economic return from dairy farms is directly dependent upon reproductive efficiency because it affects milk production and the number of calves born. There are various factors, which cause a decrease in reproductive efficiency like low fertilization rate, increased early embryonic mortality, high age at first calving, longer calving interval, and late maturity. Due to the involvement of these factors, the revenue gets affected through reduction in milk production, as well as by a lesser number of calves born. The pregnancy rate is considered to be another efficient means in evaluating the fertility of dairy animals in a herd. Further, other reproductive parameters such as service per conception, the day’s open, and calving interval are also important factors influencing reduced fertility in high producing lactating cows (Washburn et al., 2002). General and reproductive health of the animal, genetics, ovarian and endocrine functions, estrous expression, oocyte, sperm and embryo quality, oviductal and uterine environments being important physiological factors could possibly affect the fertility of a postpartum dairy cow.

10.20 Genetic control on embryonic mortality in dairy cows In spite of a large number of quantitative trait loci studies in cattle and other species, little progress has been made on the identification of major genes affecting reproduction traits (Veerkamp and Beerda, 2007). Most of the genes in early embryos are expressed in a stage-specific manner exhibiting two major patterns: starting after the onset of genomic activity or expression throughout the period before and after embryonic genome activation. This well-controlled spatial gene expression pattern has a role in the regulation of preimplantation development. There a large number genes like IFNT, FGF 2, ISG15, STAT 5A, PGR, PRL and PRLR, Osteopontin, UTMP, Integrin, Galectin 15, OAS 1, Serpina 14, etc., influencing embryonic development and its sustenance in a controlled environment. There are multiple molecular markers utilized in genotyping, including microsatellite markers, single-nucleotide polymorphisms (SNPs), amplified fragment length polymorphisms (AFLPs), and restriction fragment length polymorphisms (RFLPs). An SNP is a DNA sequence variation that occurs when a single nucleotide in the genome differs between individuals. These sequence variations have the ability to alter the amino acid produced at a specific location within a chromosome.

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10.21 Marker assisted selection Marker assisted selection (MAS) in livestock selection programs allows for increased accuracy of selection of specific DNA variations that are associated with measurable differences in economically important traits. The rate of genetic improvement achieved by MAS may be substantially greater than improvement achieved by selection based on EPD values for traits that are lowly heritable or determined postmortem. Therefore, MAS has the potential to greatly increase the efficiency of animal breeding (Davis and DeNise, 1998). Previous studies by Davis and DeNise (1998) observed that there are three phases in the development of MAS programs. First is the detection phase, followed by the evaluation phase, and finally, the implementation phase. In the detection phase, DNA polymorphisms are used as direct or linked markers in order to detect specific allele frequencies within QTL segregating populations. During this phase, markers associated with QTL are identified and the size of the QTL allele effects and the location of the QTL within the genome can be estimated. In the evaluation phase, linked markers are tested in target populations to determine whether QTL segregated within the population. Finally, in the implementation phase, predictive linked markers in a population are used within families, and direct markers are used across families in order to produce a genotypic database. These data are combined with pedigree and phenotypic information in genetic evaluation to predict individual genetic merit.

10.22 MAS versus conventional selection MAS design becomes effective if the current accuracy of selection is low, e.g., a trait with low heritability, limited, late-inlife, or after slaughter recording traits. In MAS program, the genetic gain is mainly increased by increasing the accuracy of selection. However, most current animal breeding schemes are designed such that the accuracy of selection is already high viz., accuracy of selection with 90% in the progeny testing program. This clearly indicates that MAS will be especially useful for traits where the accuracy of conventional selection is very low, such as: traits with low heritability; traits with few recording (e.g., due to expensive recording); traits that are measurable late in life, like milk production in fifth lactation or lifetime milk production in dairy cattle and buffaloes, Further, if recordings are not available at the time of selection, traits like slaughter quality traits or disease resistant traits requires expensive and risky challenge testing. Under conventional selection practices, it was observed that selection is designed to have a high accuracy of selection. If we reconsider the design of a conventional breeding scheme incorporating marker information, the result becomes more effective. For example, progeny testing has a high accuracy of selection with long generation intervals, which decreases the rate of generic gain. MAS scheme can be designed to shorten the generation interval considerably and still maintain a high accuracy of selection due to the use of marker information. If we could halve the generation interval while maintaining the same accuracy of selection, the genetic gain could be doubled. In general, the current MAS scheme consists of the two steps viz., statistically significant QTL(s) in a genome-wide scan for QTL consideration and selection for these big QTL next to selecting for a new set of polygenes. MAS nucleus scheme provides a benefit when the trait recording was before selection, e.g., the growth rate in pig or after selection, e.g., fertility trait or milk production in dairy cattle. If recording was before selection, MAS increased genetic gain by 8% in the first generation after the start of the MAS scheme. The result in terms of genetic gain was only 2% after five generations. This reveals that (i) extra response due to MAS is limited when selection for traits which are also easily addressed in conventional selection scheme; (ii) extra response due to MAS is reduced as the time period for selection program becomes longer. The second one happens as both the MAS and nonMAS schemes will fix the positive QTL allele in long term. Hence, MAS is the difference between the two schemes that will further become small in the long term. MAS opens a way to increase short term gains. This will still yield continuous benefits in ongoing MAS schemes as new QTL are being detected continuously. Accuracy of the phenotypic evaluation(s) or the performance records decides the predicting of the animal’s true breeding value or genotype (A). Further, accuracy indicates how well the evaluated phenotype for a particular trait relates to the animal’s genotype for that trait. Accuracy is a measure of the degree of confidence that we have in the estimated or predicted breeding value. Accuracy is measured on a scale of 0e1, with one indicating an exact association between the phenotypic evaluation and the animal’s true breeding value, and 0 indicates that there is no relationship between the phenotypic records and the true breeding value of the animal. Higher the accuracy, the more precisely the breeding value has been predicted. Selection accuracy helps in selecting the animals to keep as replacements for the current herd. There is phenotypic a chance of a 10%e30% increase in accuracy of selection through gene- and genome-based selection. The second component that determines the rate of genetic improvement is the amount of selection practiced, commonly called the “Selection intensity.” Selection intensity (i) is the selection differential (Fig. 10.1) expressed in terms of standard deviations (s); i.e., the ratio of (PsP0 ) over Selection. It is used in predicting genetic response due to selection because it can be related to that percent of the population saved as parents.

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FIGURE 10.1 Selection differential of a population.

The amount of genetic or true breeding value depends on variation among animals for the trait under selection (G). Genetic variation is the variability of the genotype in the herd. An inbred herd will have a low genetic variation and therefore will have a low rate of genetic change. Heritability is the proportion of the additive genetic variation to the total phenotypic variation. Heritability is important because, without genetic variation, there can be no genetic change in the population. Alternatively, if heritability is high, genetic change can be quite rapid. Using an increasing scale from 0 to 1, a heritability of 0.75 means that 75% of the total variance in a trait is controlled by additive gene action. With very high heritability, just the record of a single individual’s traits can easily be used to create an effective breeding program. The value observed while quantifying an economic trait is called as a phenotypic value of that trait. Observation in terms of means, variance, and covariance of an animal population is related to phenotypic value. The partition of phenotypic value into a component is attributable to the influence of genotype, environment, and its interaction. The genotype is comprised of an assembly of genes possessed by the individual, and the environment depicts all nongenetic circumstances that influence the phenotypic value, for understanding the properties of population with pedigree toward transmission of value in terms of genetic material from parents to offspring, which cannot be done through genotypic value alone, as parents pass on their genes, not their genotypes to the next generation. Hence, a new measure of value is required, which will refer to the gene and not the genotype. Thus, we will be enabled to assign a breeding value to the individual, a value associated with the gene carried out by the individual and transmitted to its offspring. For optimizing genetic progress (DG), the basis of selection, accuracy, selection intensity, and generation interval are key factors, which may be different in male and female. There is always a consorted/consolidated effort for maximizing DG. In dairy animals, four paths viz., sires to produce future sires (SS); sires to produce future dams (SD); dams to produce future sires (SD) and dam to produce future dams (Fig. 10.2). DG ¼ ðDSS þ DSD þ DDS þ DDDÞ=ðLSS þ LSD þ LDS þ LDDÞ

(A) A1 Young animals potential parents

Genetic Superiority above same sex in A1

Mean Age When Offspring born

(1) Bulls to breed bulls

IBB

LBB

(2) Bull to breed cows

IBC

LBC

(3) Cows to breed Bulls

ICB

LCB

(4) Cows to breed cows

ICC

LCC

A2 Second generation Potential parents

Actual parents

Bulls

Bulls

Cows

Cows

(B)

Brothers , Sisters, Cousins and etc., Grand sire Sons

Daughters

Sire progeny

The individual

Ancestors Dam

Grand dam Grand sire Grand dam

FIGURE 10.2 (A), (B) Different kinds of relatives of an individual’s upon which selection may be based. Adapted from Lasley, J.F., 1965. Genetics of Livestock Improvement, Prentice hall of India Pvt. Ltd., New Delhi.

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10.23 Whole-genome selection Whole-genome selection (WGS) is a form of MAS that utilizes genetic markers distributed throughout the entire genome so that all QTL are in linkage disequilibrium with at least one marker (Anderson, 2001; Goddard and Hayes, 2007). The objective ofn using whole-genome selection is to utilize genomic data to supplement extensive sets of performance data in order to predict genetic merit values so that producers can make informed selection decisions. An advantage of utilizing this method of selection is that it allows for the prediction of additive genetic value for epistatic and pleiotropic effects of alleles known as haplotypes for each chromosomal region that is influencing the trait of interest. Summing across all loci affecting a trait, the genetic merit of an animal can be predicted based on the multilocus genotype (Daetwyler et al., 2007). Animals with phenotypes or predicted additive genetic merits could be genotyped at a high density, with over 775,000 SNP distributed evenly throughout the genome. Either individual SNP or chromosomal regions containing haplotypes are analyzed as independent random effects under a mixed linear model to simultaneously determine genomic regions contributing to phenotype, as well as predict the additive values of each haplotype within each region. The phenotype or genetic merit of an animal can be predicted based solely upon its genotype information from predicted haplotype values (Sellner et al., 2007). In order to avoid estimating a large number of variance components for regions and to make the approach statistically tractable, Meuwissen et al. (2001) assumed equal variances associated with each chromosomal segment, as well as independence between regions. McKay et al. (2007) determined that these assumptions are violated by the existence of long-range linkage disequilibrium and because those regions closest to QTL will contribute much more variance to a trait than the rest.

10.24 Principle of genomic selection Genomic selection is based on the principle (Fig. 10.3) that information from a large number of markers dispersed across the genome can be used to detain diversity in that genome sufficient to estimate breeding values without having an accurate knowledge of where specific genes are located. For QTL identification, we perform both molecular and statistical association analyses to explain a limited fraction of the genetic variation in the population. Further, all QTLs cannot be taken into consideration if there is a lack of linkage association. Hence, an alternative procedure in which the entire genome of an individual is considered and all genes across the chromosomes explain the variation and functional effects on the traits designed to be selected. For avoiding nonadditive genetic effects, the knowledge of conventional breeding tools can also be incorporated with necessary modification to reduce generation interval.

10.25 Estimation of genomic breeding value Marker effects were estimated using the BLUP method (Meuwissen et al., 2001). The statistical model used to estimate individual marker effects was to estimate individual marker as follows: yi ¼

n X

Xij aj þ ei

J ¼1

FIGURE 10.3 Principle of Genomic Selection: Top: Prediction equation obtained from a reference population with phenotypes and genotypes; Bottom: Prediction equation used on candidates with genotypic information only. Adapted from Boichard, D., Ducrocq, V., Croiseau, P., Fritz, S., 2016. Genomic selection in domestic animals: principles, applications and perspectives. Comptes Rendus Biol. 339 (7), 274e277.

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where yi is individual record i, m is overall mean, Xij is marker genotype, aj is the random effect of the jth marker, with variance equal to the total genetic variance divided by the number of markers, and ei is a random residual. Estimation of SNP effects was performed yearly involving genomic selection, by adding the newly genotyped bulls to the reference population after they get their progeny records. Records of progenies through progeny-tested bulls (PS and CONV) or elite bulls (GS) are available at the age of 5e6 yr. The progeny test records were calculated as daughter yield deviations (DYD) directly, using the following formula vffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi u3 u s2 þ s2 t g e 1  ri DYDi ¼ TBVi 4 2 N where Ni is the number of daughters of ith sire, and ri expresses a random standard normal deviate. Hence, individual testdaughters were not simulated, and the progeny test record was assumed to be based on one record per daughter. The DYD was used to obtain more accurate BLUP breeding values for elite sire selection in PS and CONV schemes. In the PS and GS schemes, sires were added to the reference population after they received their progeny test record toward estimating marker effects. In the conventional breeding scheme, the selection steps were performed based on traditional BLUP breeding values. In genomic selection, two different strategies were implemented. For both the strategies, the male selection candidates were genotyped, and genomic breeding values (GEBV) were estimated by adding marker effects. GEBV ¼

n X

Xij aj

J ¼1

where aj is the BLUP estimate of the jth SNP effect. The accuracy of the genomic breeding values was determined as the correlation between the estimated genomic breeding values and the true breeding values (Sonesson and Meuwissen, 2009). The genomic breeding values were used for preselected young bulls for progeny testing (PS) or elite sires directly (GS) without the progeny test. Hence, genomic selection is applicable to the reduction of male generation intervals from 6 to 3 years. Further, there is a chance of an increase in the number of females born in the breeding population due to the absence of progeny testing.

10.26 Factors influencing genomic selection Irrespective of any livestock species, genetic progress depends on for parameters viz., genetic variability (A), selection intensity (i), the accuracy of evaluation (r), and generation interval (L). There is a scope to bring any change in all factors except genetic variability as the genome of a particular animal has received the genetic architectural or hereditary material from its parents. The advantage of genomic selection is that the animals can be evaluated and selected at an early age just after birth and even at an embryonic stage even without prior knowledge of its own or of progeny for any targeted trait(s). If the cost of genomic selection is lower in a large number of breeds/species and production systems, there is a possibility of a reduction in generation interval, and selection intensity will increase in reference population especially for a trait which is difficult to measure like sex-limited, meat quality, and disease tolerance. The third determinant, i.e., the accuracy of selection (r), is dependent on (i) accuracy of SNP effect and (ii) linkage disequilibrium (LD) between SNP and causal variants. Accuracy of the SNP effect depends on the reference population size (N) and heritability (h2) of the desired trait. The second factor, i.e., linkage disequilibrium, is influenced by the structure of the genome and genetic architecture of the trait. The number of independent segments plays a critical role in deciding accuracy of selection.

10.27 Present status of genomic selection in livestock breeding schemes To date, genomic selection has only been implemented in a few countries and mainly in breeding programs of Holstein cattle (Pryce and Daetwyler, 2012; Boichard et al., 2016). The first genomic evaluations were officially released for a few Holstein populations in 2009. Different strategies have since been adopted to integrate genomic selection into existing breeding programs. In other dairy cattle breeds, several difficulties have impeded the integration of genomic selection into breeding programs. First, breeding programs are generally of a smaller size than Holstein programs, which makes it more difficult to gather large reference populations. Several initiatives have, however, been created to exchange genotypes across countries, for example, the Intergenomics consortium for the Brown Swiss breed 7 Jorjani et al. (2011) and the

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collaboration between breeding schemes of the Nordic Red Dairy cattle in Denmark, Sweden, Finland, and Norway. Second, some of these breeds may have larger effective population sizes, resulting in weaker associations between markers and QTL. Both of these factors tend to reduce the accuracy of genomic predictions, which are generally lower than in Holstein populations. The recent use of high-density SNP panels was a promising option to increase GEBV accuracy in such populations. Having large densities in SNPs reduces the physical distance between markers and QTL, and hence, should strengthen the statistical association between them. At such marker density, associations between markers and QTL may also be maintained across breeds, making it possible to build across-breed prediction equations and to capitalize on reference populations of several breeds. However, preliminary analyses of high-density chips with the genomic BLUP evaluation model only resulted in marginal gains within breeds (Su et al., 2010) and across breeds. Despite these difficulties, genomic selection has been integrated into the breeding programs of a few large dairy cattle populations other than Holstein.

10.28 Prospects of genomic selection in cattle and buffaloes in India Among the breeding bulls available in cattle in India, only around 20% belong to organized breeding programs like progeny testing, and the remaining bulls are selected on the basis of the performance of dams. In the case of buffaloes, out of about 54.5 million breedable buffaloes in the country, a mere 15%e20% is bred through AI, while 80%e85% is covered by natural service. In India, the prediction equations could be made initially be based on the available records of both sire and dams having breeding values. Prediction equations using a combination of a small number of progeny-tested bulls and a large number of recorded females, the reliability of predicted breeding values of male calves could be raised up to 35%e40%. More precise prediction equations could be made if more records for both males and females are available. In a Genomic Selection model, the generation interval in the male pathway is targeted to be reduced from 6 to 6.5 years down to 1.75 years, which can result in an increase in response to selection by a factor of 2.17, in comparison to progeny testing. The costs of identifying a superior bull using genomics selection method could be reduced by more than 90% (Anonymous, 2017). The impact of genomic selection is expected to be more pronounced in indigenous breeds of cattle as the selection of males at present is solely based on the mother’s yield, and the accuracy of selection of bulls is very low.

10.29 Genetic gain by genomic selection The success of the genomic selection is based on increased genetic progress on both the bull and cow genetic pathways by reducing costs than conventional selection schemes, but also by the potential of using this rich source of information to manage genetic resources. Selection differential(s) (SD) and generation interval(s) (GI) were calculated based on four-path selection model, which includes sire(s) of bulls (SB), sire(s) of cows (SC), dam(s) of bulls (DB), and dam(s) of cows (DC) pathways. After the implementation of genomic selection, significant reductions were observed in GI, especially the SB and SC pathways. GI in the SB path reduced from 7 years to less than 2.5 years, and the GI of DB path declined from about 4 years to nearly 2.5 years. Further, through this path, modest genetic gains were observed. Response to selection was significantly influenced due to the genomic approach for lowly heritable traits like DPR (daughter pregnancy rate), PL (productive lifespan), and SCS (somatic cell score). There was a significant change in genetic trend from close to zero to large and favorable, resulting in rapid genetic improvement in fertility, lifespan, and health traits. This clearly indicated that genomic selection contributed significantly toward genetic improvement in dairy cattle in the USA. Application of genomic selection through the four-path selection model revealed that the genetic gain per year increased from 50% to 100% for milk yield traits and from three to four-fold for lowly heritable traits. Daughter pregnancy rate (DPR) is the measure of the ability of the daughter of a bull to become pregnant and receive 11% of the emphasis on lifetime total merit. The gradient of positive direction represents the higher-level fertility in cows. On account of being a lowly heritable trait, there is a limited scope of improvement through conventional breeding. It remains as a negative gradient throughout 30 years of selection as there is genetic antagonism in milk yield and fertility. There was a steady increase in the selection differential of this trait through SC and DB paths. Young animals selected for breeding on the basis of their parents’ performances and themselves are regarded as the potential parents of the next generation. The decision as to which of these animals actually will pass their genes on is made on the basis of some measurements of their own genotype by own performances in cows and by progeny tests in bulls. Genes may be transmitted to the next generation in four ways, which are diagrammatically represented as follows.

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10.30 Methods of genomic selection The presence of a large number of markers, and the effect of each marker on quantitative trait is the key factor for evaluation of genomic breeding value. to determine breeding value, using suitable statistical models, either direct effect of markers is exclusively used or covariance model using marker information is considered. BLUP (Best linear unbiased prediction), GBLP (Genomic best linear prediction) is used in which breeding values are estimated based on relationship matrix with marker information instead of pedigree information. In GBLP all markers carry equal weightage. This model ignores the true genetic merit of the trait. Further, the covariance of genomic breeding values of two animals is proportional to their proportion of genome share. The Bayesian method of evaluation is also used to calculate the genomic breeding value in which larger weights are provided to SNPs potentially closer to causal variants. This method is important multi marker QTL mapping.

10.31 Advantage of genomic selection over conventional breeding Literatures revealed that for lowly heritable traits, genomic selection yields higher genetic progress than conventional selection. Further, thePS scheme exhibited higher accuracy for the genomic breeding values than the GS scheme, except when heritability was 0.01 (Table 10.2). For all heritability higher than 0.01, accuracy was increased with the number of selected sires within the GS scheme was increased, but it could not compensate for the reduced selection intensity, hence, genetic progress enhanced with a lower number of sires selected. However, the reduction in the inbreeding rate was higher than the reduction in genetic gain when the number of sires was increased. With an increase in the number of sires, there could be a decrease in the rate of inbreeding. Therefore, GS schemes provided the highest genetic gain, even when schemes were compared at the same rate of inbreeding (Lillehammer et al., 2011).

10.32 Reabilities (r2v) of EBV and regression coefficient (REG) of corrected phenotypic values Regression coefficients estimated out of 115 sires genotyped in Brazil revealed serious bias of prediction for milk and fat yield using pedigree information. By using ssGBLUP biasedness was corrected by adding genotype information. In comparison to corresponding pBLUP, there was a decrease in reliability and an increase of bias in ssGBLUP was observed for fat yield using the Brazilian population and for protein yield using three populations. Reliabilities (r2v) of genomic predictions from both the traits using pBLUP and ssGBLUP increased significantly than conventional methods of prediction based on phenotypic data alone (Table 10.3). The genetic gain using data of bulls originated from Nordic and French was observed to be much higher than the gain for nongenotyped Brazilian cows. These gains in reliabilities were consistent with previous studies on improvement of genomic prediction based on reference populations in a consortium of countries, like Holstein in the Euro Genomics (Lund et al., 2011) and North American (Van Raden et al., 2009) consortia, and for the Brown Swiss breed from Intergenomics consortium (Zumbach et al., 2010).

10.33 Genomic evaluations in developing versus developed countries In developing countries, especially in Asia and Africa, most of the production protocols fall under in small-holder systems, which are characterized by small herd sizes, without having proper performance and pedigree records. Hence, there is an absence of conventional genetic evaluation systems. In India, the National Dairy Development Board, along with the Ministry of Agriculture and Farmers Welfare, Government of India, had already taken up progeny testing and genomic selection program in indigenous breeds of cattle. Similarly, in some countries like Brazil in Latin America, due to action of breeders associations, there is the establishment of some degree of data and pedigree recording and genetic evaluation system although there is still a lack of full set up of modern breeding structures such as AI companies, to drive breed improvement programs further in a massive way (Silva et al., 2016; Boison et al., 2017). In the current genomics era, most of the programs for genotyping in developing countries like Brazil were undertaken by breed organizations or breeders’ associations, and development projects like East Africa Dairy Development Project, and the African Dairy Genetic Gains Cattle project (Carvalheiro et al., 2014; Brown et al., 2016; Silva et al., 2016). There is a constraint that includes the limited number of genotyped animals, mostly females, which influences both the size and structure of the reference and validation populations. In developing countries, due to small data set of genotyped individuals, along with either no or less complicated conventional genetic evaluation systems have resulted in the implementation of GBLUP and various Bayesian methods (Table 10.1). GBLUP, which has been commonly utilized

TABLE 10.1 Summary of genomic selection in developing countries.

Response variable

Accuracy of prediction

448 (fivefold cross validation)

BayesC

EBVs

0.34e0.58

0.40e0.87

264-281 bulls only or plus 1177 e1597 cows

115e152 bulls

GBLUP

dEBVs

0.46e0.56 (only bulls reference) 0.47e0.62 (bull þ cow reference)

0.73e0.97 (bulls only or bulls plus cows in reference)

1038 Cows

565e835 Cows

178e448 Cows

GBLUP and BayesC

Corrected phenotypes

0.32e0.41 (GBLUP) and 0.28e0.35 (Bayes C)

lllumina 50K (cows) and lllumina HD (sires)

113 sires 3545 cows

2765

691(fivefold crossvalidation)

GBLUP, BayesC and ssBLUP

dEBV

0.38e0.48 Validation set by k means clustering 0.40e0.60 ( validation set by random clustering

0.29e1.46 (K-M) 0.25 e0.83 (random)

Heifer re breeding age at first calving, early pregnancy occurrence

lllumina HD

2056 Females

1853

185(10-fold cross)

GBLUP, BayesC and IBLASSO

dEBV and corrected phenotypes

0.29e0.54 (GBLUP) 0.34 e0.57 (BayesC) and 0.37e0.58 (IBLASSO)

0.84e0.88 (GBLUP) 0.89e1.14 (BayesC) and 0.81e0.87 (IBLASSO)

REA, BFT, and HCW

llllumina HD

1756 Nellore steers

1405

351(fivefold cross validation)

Bayesian ridge regression (BRR) BayesC (BC) and Bayesian lasso (BL)

Corrected phenotypes and EBVS

0.21e0.46 (ERR) 0.23e0.46 (BC) 0.22e0.47 (BL)

0.40e0.99 (ERR) 0.37 e0.93 (BC) 0.39e1.02 (BL)

Population size

Reference population

References

Breed

Traits studied

Chip size

Boddhireddy et al. (2014)

Nellore cattle

22 traits composed of reproductive, productive, visual body conformation scores traits

lllumna BovineHD BovineSNP50 (50K) Chip

2241

1793

Boison et al. (2017)

Gyr (Bos indicus) dairy cattle

Milk, fat, and protein yields and age at first calving

lllumina bovineHD (Bulls) Bovine SNP50(50K) chip (cows)

464 bulls 1688 cows

Brown et al. (2016)

East Africa cross bred cattle

Milk yield

lllumina bovine HD

Cardoso et al. (2014)

Bradford and Here ford cattle

Tick resistance

Costa et al. (2014)

Nellore cattle

Fernandes Ju´nior et al. (2016)

Nellore cattle

Size of validation set

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Models

Regression coefficients (response variable on predictions)

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Continued

Reference population

Size of validation set

Regression coefficients (response variable on predictions)

Response variable

Accuracy of prediction

GBLUP, BayesC, BLASSO

dEBV

0.17e0.72 (GBLUP) 0.20 e0.69 (BayesC) 0.19e0.74 (BLASSO

0.75e1.24 (GBLUP) 1.01e2.35 (BayesC) 0.91e2.17 (BLASSO)

91e337

Ss GBLUP, GBLUP and Bayes C

Corrected phenotypes and EBVs

0.23e0.48 (GBLUP) 0.23 e0.48 (BayesC) 0.30e0.49 (BLASSO)

0.78e0.80 (GBLUP) 0.05e3.10 (BayesC) 0.75e1.16 (BLASSO)

531(GBW) 246(GWY)

GBLUP

dEBV

0.21e0.48 (GBV) 0.21 e0.28 (GWY)

0.38(GBW) 0.25(GWY)

References

Breed

Traits studied

Chip size

Population size

Neves et al. (2014)

Nellore cattle

15 economically important traits

lllumina HD

691 bulls

307e494

115e187

Silva et al. (2016)

Nellore beef cattle

RFI, FCR, ADG, and DMI

lllumina HD

788 cows

424e617

Terakado et al. (2014)

Nellore cattle

Weight gain birth to weaning (GBV) and weaning to yearling (BWY)

lllumina HD

1658 females and 1002 males

2118(GBW) 988 (GWY)

Models

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TABLE 10.1 Summary of genomic selection in developing countries.dcont’d

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TABLE 10.2 Rate of inbreeding (DF), Genetic gain (DG), and Accuracy (Acc) of genomic breeding values with wide range of heritability (Lillehammer et al., 2011). h2 [ 0.01 DF2

Breeding scheme

h2 [ 0.05

DG2

Acc.3 ..

DF

h2 [ 0.30

DF

DF

Acc

DF

Acc

conv

1.0

1.0

1.0

1.0

..

1.0

1.0

..

PS_125_260

0.76

1.20

0.51

0.69

1.15

0.64

0.57

1.11

0.72

PS_60_542

0.66

1.26

0.54

0.62

1.14

0.64

0.67

1.09

0.69

GS_12

0.64

1.70

0.60

0.93

1.40

0.61

1.14

1.29

0.60

GS_40

0.27

1.44

0.57

0.33

1.24

0.62

0.35

1.17

0.63

4

TABLE 10.3 Reabilities (r2v) of EBV and regression coefficient (REG) of corrected phenotypic values on EBV for 115 Brazilian bulls (105 bulls for protein yield) (Li et al., 2015). BRA pBLUP 2

BRA-NOR ssGBLUP

pBLUP

BRA-(NORDFRA)

ssGBLUP

pBLUP

ssGBLUP

Trait

(r v)

REG

(r2v)

REG

(r2v)

REG

(r2v)

REG

(r2v)

2

REG

(r v)

REG

Milk Yield

0.240

0.775

0.263

0.819

0.338

0.831

0.383

0.938

0.408

0.934

0.418

1.076

Fat Yield

0.156

0.728

0.133

0.653

0.169

0.674

0.278

0.884

0.278

0.792

0.313

0.992

Protein Yield

0.200

0.835

0.266

0.965

0.369

0.930

0.382

1.007

0.448

1.017

0.433

1.143

BLUP, pedigree based BLUP; ssBLUP, Single-step genomic BLUP, BRA, Brazilian Holstein populations; BRA-NOR, Brazilian and Nordic Holstein populations; BRA, (NOR þ FRA), Brazilian, Nordic and French Holstein populations.

with G, usually was computed according to the method evolved by Van Raden (2008). Computation of G has enabled the estimation of genetic relationships between different groups of animals and to undertake genetic evaluations in the absence of pedigree information (Mrode et al., 2018). The use of ssGBLUP was carried out when the availability of genotypic information was only on a limited proportion of animals (Misztal et al., 2009). Further, Cardoso et al. (2014) and Silva et al. (2016) calculated genetic merit using both pedigree and genotypic information, which resulted in higher accuracy. Bayesian methods were utilized, especially with limited data size, yet no clear advantage of these methods over GBLUP or ssGBLUP was demonstrated. The basis of faster genetic progress from a genomic selection in developed countries was executed by routine genomic predictions several times in a year. Although several studies were carried out in different breeds in developing countries, the routine genomic prediction was undertaken in a limited number of breeds (Table 10.1). In Nellore beef cattle in Brazil, various parallel breed improvement programs were implemented, and recently, the genomic selection was incorporated in some of these breeding programs (Carvalheiro et al., 2014). Further, it is indicated that several independent Nellore breeding programs were already developed toward prediction equations for usual and difficult/ expensive to measure traits; however, some of the programs using genomic predictions were more a marketing tool than a selection tool. Carvalheiro et al. (2014) summarized the two commercial models driving GS in the Nellore cattle. In the first scenario, it is narrated that the breeders who cannot carry out genotyping and genomic prediction equations are dependent on multinational private companies, and intellectual property remains with them as they invested in their breeding and development program. Under this model, the genomic breeding values (GEBVs) are calculated by combining genomic predictions and regular EBVs as correlated traits in a multitrait mixed animal model analyses (Garrick, 2011). Therefore, the sustainability of the programs depends on the interest of the commercial companies that invest in recalibrating the prediction equations.

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The second model exhibited breeding programs and the breeders who have full access to the genotypes. It was considered to be a very attractive model because no dependencies prevail in any of two segments, enabling breeding programs to change their service providers without any issue if they are not satisfied with genotyping cost or with the quality of the genetic evaluations. In Tanzania and Ethiopia, the Africa Dairy Genetic Gains (ADGG) projects recently established a foundation for routine genetic evaluation using the genomic relationship matrix along with screening and selecting young bulls using genomic predictions. The nonexistence of AI companies to drive genetic improvement programs, indicated that genetic, as well as genomic evaluations, were inevitably linked to either National Artificial Insemination Centers or breed societies to deliver the superior germplasm.

10.34 Genomic selection in developed countries Genomic selection in developed countries was incorporated into breeding schemes using two different ways, viz., genomic preselection (PS), and genomic juvenile (JS) schemes. The PS scheme involves using GEBVs to preselect young males for progeny testing (PT) programs. Further subsequent steps for the selection of males remain the same as in PT schemes. In comparison to PT schemes, through PS schemes, there is an enhancement of accuracy of selection in young males incorporating their genotypic information. In JS schemes, the use of genomic information is more detailed. Although genomic breeding values of young sires are less accurate than conventional breeding values estimated in progeny-tested bulls, the loss of accuracy of selection is compensated by a tremendous reduction in generation intervals by avoiding using progeny testing. In both the schemes, females are genotyped to increase accuracy, as well as intensity toward the selection of females. Genotyping both males and females is favorable in the genomic selection of dairy cattle breeding schemes (Sorensen and Sorensen, 2009). Phenotypic information of a targeted test trait genetically correlated with the selected trait and recorded on a large set of data can be integrated in genomic evaluation model to improve the accuracy of predictions for traits with low heritability when the genetic correlation between both traits is large (Calus and Veerkamp, 2011; Buch et al., 2012). GEBV reliabilities larger than 0.5 to 0.6 are already achieved in the homogeneous dairy cattle populations with large reference Holstein populations for traits with moderate to high heritability. In breeds with large effective sizes or small reference populations, the reliability of GEBVs is generally lower (0.5) even for production traits.

10.35 Genome-wide signatures for selection using molecular genomic tools 10.35.1 SNP Chip The most efficient method for genotyping large numbers of SNPs is through the design of a high-throughput assay that includes a large number of SNPs. These high-density panels are referred to as “chips” and are a valuable resource for genetic studies in livestock. Some of these studies include genomic selection for economically important traits, QTL identification, comparative genetic studies, and parentage testing. High-density SNP genotyping has become a reality as a biomedical diagnostic for predicting predisposition to heritable genetic diseases. Similar applications of this technology opened a new choice in cattle breeding for improvement of herd health, increased animal productivity, and increased selection accuracy. Currently, there are multiple platforms available for use in whole-genome association studies. These technologies utilize probe labeled primers in order to distinguish the two alleles of an SNP. For utilizing this technology effectively, it is important to determine the chromosomal position of the SNP of Interest well in advance (Schmitt et al., 2010). The chip itself contains over 700,000 SNP markers uniformly distributed across the genomes of various cattle breeds (Illumina Inc., San Diego, California). Genome sequencing and SNP genotyping technologies, in addition to modern bioinformatics and statistical tools, have brought a transition from studies focusing on the analysis of neutral variation to functional variation. These recent developments resulted in the discovery of new tools to address the fundamental and applied questions in evolutionary and developmental biology, as well as in animal breeding. Genomic Selection for Cattle in Developing Countries with whole genome approaches toward development of SNP Chip sets has led to studies on identification and mapping of genes and QTLs, genome-wide association analysis (GWAS) and genome-wide signatures of selection, introgression, and/or admixture etc. The studies involved the identification of many genes, and out of which, some were incorporated into selection schemes. In developed countries, whole genome sequence analysis and GS are being applied in breeding schemes of major food animal’s viz., cattle, sheep, goats chicken, and pigs. In developing countries, genomic tools are applied to assess the genetic diversity and admixture and signatures of selection to identify genomic regions and variants contributing to a variation. The use of genomic tools revealed that indigenous cattle in developing countries have wide genome diversity

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than commercial breeds (Kim et al., 2017). Further, Kim et al. (2017) also reported that the genomes of indigenous breeds are admixed, which suggests genomic diversity as an efficient adaptation strategy. SNP genotyping and whole-genome sequencing have shown the genome admixture is of ancient and recent origin. Studies on zebu cattle from Kenya, Uganda, and Nigeria exhibited an even admixed autosomal Asiatic indicine_African taurine genome composition along with European taurine ancestry (Mbole-Kariuki et al., 2014; Bahbahani et al., 2017; Hanotte et al., 2002; Decker et al., 2014). Literatures revealed that Asian indicine African taurine germplasm composition was ancient (Hanotte et al., 2002; Decker et al., 2014) while the European taurine background was originated from crossbreeding of local cattle with European Bos taurus breeds. Based on research findings, it was revealed that Borgou cattle of West Africa is a stabilized admixed breed having genetic contributions by four African taurines (Baoulé, Somba, Lagune, N’Dama) and two African Zebu (Fulani, Bororo) cattle, whose origin was traced back to about 130 years ago (Flori et al., 2014). According to Kim and Rothschild (2014), genomes of Kenyan local cattle were contributed by several B. taurus breeds viz., Guernsey, Norwegian Red, and Holstein, in which, the contribution of Holstein-Friesians was the most significant. The authors postulated the admixture happened in recent times. Admixed genomes are also a common feature of indigenous and locally developed breeds of cattle in South Africa (Makina et al., 2014). Admixed genomes were also observed in Asian (India, Pakistan, China, and Indonesia) Bos indicus cattle, which show evidence of Bos javanicus ancestry (Decker et al., 2014). Kumar et al. (2003) revealed that an ancestral influence from taurine cattle in South Asian Bos indicus cattle, probably of Near-Eastern origin. Southeast Asian indicine ancestry was observed in the genomes of Thailand cattle (Wangkum hang et al., 2015). It is evident that there is a lack of availability of written pedigree records in most of the small-holder farms in developing countries, which makes it difficult to make breeding decisions. Genomic technologies could be valuable in this case in assessing breed composition and parentage assignment (Werner et al., 2004; Weerasinghe, 2014). Recently, Strucken et al. (2017) demonstrated the application using crossbred cattle in East Africa (Kenya, Uganda, Ethiopia, and Tanzania). The authors used two marker panels with 200 SNPs each. One panel estimated the best dairy breed compositions, and the other determined the accurate estimates of parentage lineage. A composite panel using 400 SNPs provided sufficient accuracy toward estimating breed admixture proportions but not parentage identification. The invention of new technologies assessing genome architecture with a high resolution like full genome sequences, HD Chips, etc. has resulted in a large number of studies investigating genome-wide signatures of selection in indigenous cattle in developing countries and especially in African cattle contributed better result more precisely. In a study of 18 candidate regions under selection and intersecting genes and QTLs associated with production and reproduction performances along with adaptation to environmental stress like immunity and heat stress were determined in East Africa cattle for analysis of SNP genotype datasets (Bahbahani et al., 2017). Further, they stressed upon several QTLs related to dairy traits, which overlapped candidate selection regions in Kenana and Butana cattle breed using the analysis of SNP genotyped data. Using whole-genome scans, Gautier et al. (2009) identified 53 genomic regions comprising 42 genes having functions related to immune response, nervous system and skin, and hair properties in West African cattle. In another investigation, 47 candidate regions spanned genes associated with adaptation to tropical environments, nervous system, immune response, production, and reproductive performances in South African cattle were reported by Makina et al. (2015). Genome sequences studied in indigenous breeds of cattle originated from East, West, and Southern Africa revealed by Kim et al. (2017) determined signatures of selection including genes and/or pathways controlling anemia, feeding/drinking behavior and circadian rhythm in the N’Dama, coat color and horn development in Ankole, and heat tolerance/thermoregulation and tick resistance in Boran, Ogaden, and Kenana cattle. Results of selection signature studies involving genes with specific functions related to production, reproduction, and adaptation, suggested that genomes of cattle African indigenous cattle were uniquely selected to maximize hybrid fitness for adaptation to reproduce and perform in stressful environments.

10.36 Functional genomics in fertility traits Based on functional genomics studies with the genome-wide association for fertility and production traits in dairy cattle, it was observed to study SNPs located within or close to 170 candidate genes. Data from 2294 Holstein bulls were genotyped for 39,557 SNPs, which exhibited a total of 111 SNPs on chromosomal segments of a candidate gene. Allele substitution effect for each SNP was calculated using a mixed model with a fixed effect of the marker and a random polygenic effect. Results from the analysis with the kinship matrix built from marker genotypes were reported to be more conservative than the analysis with the pedigree-derived relationship matrix. Results depicted that out of 16 SNPs having significant effects on both kinds of traits, 10 provided evidence of an antagonistic relationship between productivity and fertility. However, it was found that four SNPs had favorable effects on fertility, as well as on production traits, one SNP with favorable effects on

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fertility and percentage traits, and one SNP with antagonistic effects on two fertility traits. Previously, most quantitative genetic studies indicated genetic antagonisms between production and functional traits, improvements in both production and functionality might be possible when focusing on a few relevant SNPs. Hence, investigations combining input from quantitative genetics and functional genomics with association analysis could be applied for the identification of such SNPs.

10.37 Approaches for developing disease tolerant livestock Infectious disease is a major challenge to livestock breeders. In the livestock production system, economic losses due to infectious diseases are accounted for around 20% in developed countries and 35%e50% in developing countries, respectively. However, the true cost of disease occurrence is a complex phenomenon. There are many zoonotic diseases like Tuberculosis, rabies, brucellosis, anthrax, avian influenza, etc. are important from both human and animal health are concerned. The incidence of infectious diseases impedes the growth of livestock industry, despite the widespread application of antibiotics, vaccines, quarantine, and other conventional disease control measures. Control of infection in livestock is genetic disease resistance, the inherent capacity of a previously unexposed animal to resist a virulent challenge. Genomics has opened a new avenue in disease investigation and vaccine research in livestock. So far, genetic selection of animals placed emphasis on productivity and efficiency, which has potentially reduced natural disease resistance. It is now appropriate to implement these combined efforts of genomics with conventional breeding practices for the selection of superior livestock to bring cost-effective and faster genetic improvement. Classical genetic selection, recently aided by genomic selection tools, has been successful in achieving remarkable progress in livestock improvement. However, genetic selection has led to decreased genetic diversity and, in some cases, acquisition of undesirable traits. The selection of superior dairy animals based on true genetic merit or breeding value is the main key for their genetic improvement. Genetic improvement depends on two principal components viz., (a) the existence of variability in the population (b) the population which heritable, i.e., the heritability. Success for selection also depends on having clear, well-defined objectives, which support selection through replacing less desirable alleles by better alleles in the population. The genetic progress is the product of the intensity of selection times the heritability of the trait under selection times the phenotypic standard deviation of that trait. If any of these three parts is low, genetic progress through selection will be slow. The economic value of the trait may still justify efforts to improve it through selection, as such improvement is a permanent change that benefits all future offspring. Heritability helps the dairy producer to decide which traits justify improvement through selection. Heritability is one important component of the equation used to predict genetic progress from selection to improve a trait.

10.38 Candidate genes for disease resistance for milk production Farmers prefer disease-free dairy cattle due to a couple of reasons, caused by problems associated with reproduction, poor milk quality, low milk production, lameness, or a number of other possibilities. Literatures revealed that 26.7% of cattle were culled due to poor reproductive performances. Dairy farmers having a good reproductive program creates an optimal dairy environment, reducing losses due to income caused by long days open and reducing reproductive culling. Economic analysis shows that calving intervals extended beyond 13 months result in the reduced annual return. Studies exhibited that high somatic cell counts (SCC) influence reproductive efficiency indirectly. Cows having postcalving metabolic disorders are prone to long-term damage to the reproductive tract which results in infertility problems. Damage to the uterus happens during a difficult calving. Sometimes, damage can be too critical to repair and can cause future reproductive problems. Higher the number of services per conception, the less fertile is the animal. Each day cows stay open past the voluntary waiting period costs the dairy producer’s income.

10.39 Production of disease-resistant genetically modified livestock Disease control strategies mainly focus on either the pathogen or the host. Conventionally, control of animal disease involves either destruction of the pathogen or the vector carrying the pathogen, i.e., through vaccination, treatment using antibiotics and other drugs, or spraying swamps with dichlorodiphenyltrichloroethane to kill mosquitoes. Despite the success of these conventional strategies in combating specific diseases, there are many continuing challenges relating to animal health and disease. Many previously used control strategies, i.e., the use of antibiotics is cumbersome from health and legal point of view. Further, due to the indiscriminate use of antibiotics, antihelmintic, etc., pathogens get resistant, and the genome get mutated and evolve in such a way that the control strategy is no longer effective.

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As an alternative to conventional approaches, powerful genetic tools are becoming available to assist in combating disease. Animals were selected on observable phenotypes without understanding the molecular mechanism of inheritance. With the advent of molecular tools along with advanced animal breeding and computational tools, desirable genotypes controlling the economic traits of farm animals can be selected for genetic improvement. Genetic engineering of livestock is used for producing animals with disease resistance. Presently, it is possible to modify livestock genomes to block the expression of endogenous and exogenous genes like a viral infection. In the conventional breeding process, for selection of desirable characters like growth, production, etc. some undesirable traits viz., poor reproductive performance and lower resistance against disease are automatically incorporated. In addition, QTLs present in the parents are transmitted in the next generation for the selection of superior progenies, which severely limits the range and extent of genetic improvement. To overcome these limitations, gene addition through transgenic technology potentially opens a new vista. Genetic modification offers alternative strategies to conventional animal breeding. This technology can be applied for a specific application, where genetic variation does not exist significantly in a population toward a holistic genetic improvements program. With this approach, there is a possibility to enhance the ability of the animals to mount an appropriate immune response against the pathogens or to generate an effective system that would directly block the entry of pathogens and destroy them. Further, a combination of strategies may prove to be the most successful approach. The potential of GM technology toward the genetic improvement of novel economic traits of livestock can be generated. There are two advantages of using genetically modified (GM) livestock in our society. First, the GM animals could be used for a model for studies on disease progression and its control. In this situation, it is likely that relevant information related to human disease may also be gathered indirectly. Second, GM-based disease-resistant livestock and their produce could be used in the food chain. In this scenario, the introduced trait would not be normally found in nature; the aim, however, would be to mimic or interfere with specific aspects of the infectious agent. There are various possible strategies, as listed below, and the authors anticipate that current efforts in disease biology will enable more to be devised; for some strategies, there is preliminary data to encourage further exploration of this use of GM technology:

10.39.1 Dominant negative proteins Mutated key factors in pathogen infection, like cell surface receptors, can easily block disease progression. Interaction of parasite with surface molecules on mammalian host cells initiates host cell responses, which significantly influences the progress of the infection. The Abl family of protein kinases and also PKR are nonreceptor protein kinases that play an important role in determining the outcome of Leishmania infections.

10.39.2 Ribonucleic acid interference (RNAi) This method relies on the ability of specific short RNA sequences to anneal with the RNA of the pathogen, causing the destruction of the foreign RNA. RNAi requires access to the target RNA, which may limit this approach to viruses. Mechanism of silencing gene expression through RNA interference (RNAi) toward activating the RNAi pathway is useful for its application to enhance livestock production through increased production efficiency and prevention of disease. Use of RNAi technology to study the role of oncogene expression in leukemia cell lines and to validate the therapeutic potential of RNAi for treating blood disorders.

10.39.3 Ribonucleic acid decoys Short strands single-stranded RNA can mimic mRNA sequences and compete for proteins that act as translational activators and stabilize mRNA. These RNA decoys are potentially attractive and useful. Expression of RNA sequences, which mimic specific sequences within a pathogen, can disrupt the activity of the pathogen’s replication machinery. Again, this approach is probably restricted to specific viruses like influenza causing virus. Role of both short and long noncoding RNAs (ncRNAs) for the regulation of gene expression has drawn significant attention in recent years. Noncoding RNA-based regulation is achieved through a variety of mechanisms in which some are relatively well understood, while others are not much clear. MicroRNAs (miRNAs) being endogenous small RNAs regulate gene expression in eukaryotic organisms. Recently, it is observed that role for long ncRNAs i.e. miRNA decoys, also referred to as target mimics or sponges in which long ncRNAs carry a short stretch of sequence sharing homology to miRNA-binding sites in endogenous targets. As a result, miRNA decoys are able to sequester and inactivate miRNA function. Engineered miRNA decoys are also efficient and useful tools for studying gene function.

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10.39.4 Animal pharming The use of transgenic animals to produce human drugs attracts a lucrative world market due to serious unresolved human and animal health issues. Transgenic animals are those animals that are genetically transformed by splicing and inserting a foreign gene of human and animal origin into their chromosomes. This inserted gene further successfully enables an animal to make a certain pharmaceutical protein in its milk, urine, blood, sperm, or eggs, or to grow rejection-resistant organs for transplant. In 1985 first transgenic farm mammal viz., a sheep called “Tracy” was produced. Tracy had a human gene, which expressed high levels of human protein, i.e., alpha-1-antitrypsin. Without this protein, there is a chance of a situation that may lead to a rare form of emphysema. Through transgenic milk production, there is a scope of a cost-effective system for the manufacturing of large amounts of complex proteins like recombinant human antithrombin (rhAT) expressed in transgenic dairy goat. In this process, in general, goat expresses rhAT in their milk at approximately 2 g/L. The human AT purified from milk is structurally indistinguishable from human plasmaederived AT with the exception of carbohydrates. For clinical use against deep-vein thrombosis, recombinant antithrombin protein would be useful. Due to the problem of diabetes disease worldwide, there is a steady increasing demand for insulin. Current insulin manufacturing capacities are incapable of meeting the requirement. The feasibility of producing human proinsulin in the milk of transgenic animals was attempted. Through this experiment, four lines of transgenic mice harboring a human insulin cDNA with expression driven by the goat b-casein gene promoter were generated. The expression level of human proinsulin in milk was recorded as high as 8.1 g/L. A higher level of expression of this transgene was detected in the mammary gland of mid-lactating animals than early and late lactations. The level of blood glucose and insulin and the major milk compositions were unchanged, and the transgenic animals did not exhibit any health defect. The mature insulin derived from the milk proinsulin remains biologically active.

10.39.5 Antibodies Transgenic production of antibodies in the host animal may act in an analogous manner to vaccination. The use of recombinant proteins has steadily increased during the last 2 decades. A large number of human proteins and potential therapeutic targets and their development for therapeutic uses were identified. The clinical application often requires a large amount of highly purified molecules for multiple or chronic treatments. The development of very efficient expression systems has been the key to the full exploitation of recombinant technology. Human monoclonal antibodies have significant applications for human therapy. One of the most promising approaches to produce therapeutic human monoclonal antibodies is the creation of a mouse strain engineered for the production of large quantities of human antibodies in the absence of mouse antibodies. Recently, such mice are being produced by introducing segments of human immunoglobulin loci into the germlines of mice deficient in mouse antibody production as a result of gene targeting. These mice are able to produce significant amounts of fully human antibodies with a diverse adult-like repertoire. Further, immunization with antigens generates antigen-specific fully human monoclonal antibodies. Such strains of mice provide the optimal source for producing human monoclonal antibodies with high affinity and specificity against a broad spectrum of antigens, including human antigens.

10.40 CRISPR Highly specific targeted CRISPR-Cas9 is an editing tool is useful in many applications in the livestock industry. Using an engineered nuclease deficient Cas9 enables the redesigning the system for targeting genomic DNA without cleaving it. It was discovered that the coexpression of Cas9 without endonuclease activity and gRNA created a DNA recognition complex, and this complex acted upon toward interfering with the transcriptional elongation (Makarova et al., 2006). The efficacy of dCas9 for sequence-specific gene suppression was first established in E. coli. This high throughput tool is called CRISPR interference. By pairing dCas9 with a sequence-specific sgRNA, the dCas9: sgRNA complex enables to interfere with the transcription elongation by blocking RNA polymerase. It can also prevent transcription initiation by disrupting transcription factor binding. The CRISPR method is highly useful in suppressing genes. It is specific and multiplexable, but along with these benefits, dCas9 may repress downstream genes within an operon instead of an individual gene (Dominguez et al., 2016).

10.41 RNA editing In addition to double-stranded DNA and RNA sequences used for gene editing by the CRISPR-Cas9 system. RNA editing CRISPR-Cas9 system composes of a PAM presenting DNA oligonucleotide (PAMmer), ssRNA (single strand RNA),

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gRNA, and a Cas9 protein. The PAMmer functions as a PAM, which is specifically documented by Cas9 and directs Cas9 to bind or cleave the target ssRNA. The 50-extended PAMmers, which contain several ssRNA-matched bases and immediately lead the PAM, are required for the specific binding of the target ssRNA. Furthermore, dCas9-gRNA has a higher affinity for binding the target ssRNA than wild type Cas9 (O’Connell et al., 2014). As RNA has a different function from DNA, it could make the CRISPR-Cas9 system more flexible than other applied genome-editing tools.

10.42 Disease treatment Bacteria derived CRISPR-Cas9 systems to influence mammalian genomes presents huge opportunities toward the curing of human diseases (Doudna and Charpentier, 2014). According to a report by the National Institute of Health, there are thousands of human diseases, but only 500 have treatment (Mout et al., 2017). Many thousands of diseases are caused with gene alterations in genome. CRISPR technology enables correcting such genetic alterations, making a large number of diseases therapeutic targets. The development of new animal models, as well as cellular models for disease-related studies, have greatly benefitted from the application of the CRISPR Cas9 system. Several self-governing studies have been successfully removed the mutation in the dystrophin protein responsible for the most common form of Duchenne’s muscular dystrophy (Dmd) by removing the mutated Dmd exon 23 of the gene via adeno-associated virus-9 (AAV9). The CRISPRCas9 system has also been used in correcting cataracts in mice by injecting zygotes with the CRISPR-Cas9 system with the specific gRNA for the mutant CRYGC gene allele. Pig models for Parkinson’s disease were generated via the CRISPRCas9system by targeting genes for DJ1, Parkin, and Pink1. CRISPR-Cas9 was applied to knock out the p53 gene to generate p53 biallelic mutant monkeys (Liang et al., 2015). Targeting the cleavage of the b-globin gene (HBB) in human tripronuclear zygotes was accomplished using the CRISP-Cas9 system. All the above-mentioned studies highlighted the need to improve the reliability and specificity of the CRISPR-Cas9 approach before attempting clinical applications.

10.43 Conclusion The success of the Genomic Selection (GS) method critically depends on the availability of a large database of phenotypes and genotypes. GS also appears to be a promising tool to curb inbreeding rates because it allows the exploitation of withinfamily genetic variance from the earliest selection stages. However, to be effective, breeders must consider using a diversified panel of genomically evaluated bulls, both to mitigate the risk of using bulls with poor merit and limiting the erosion of genetic diversity. In India, there is an urgent need and requirement of the use of genomic selection for the genetic improvement in livestock, especially in large animals. The actual genetic gain could be reaped by its usage for genetic improvement of indigenous cattle where progeny testing is not possible in the near future under Indian conditions. Among several tools and approaches available to investigate the quality and or functions of the above components, examining them at a molecular level using gene expression profiles is a necessary contemporary research. Vast numbers of genes are identified, and their temporal and spatial expressions in different reproductive tissues have been studied in mammals, including bovines. Several of those genes are implicated from the developmental or functional point of view for fertility in embryos, CL, and endometrium. Analyzing the gene expression levels of those important genes at specific stages reveals the developmental or functional capacities of those tissues. The factors influencing fertility in dairy cows such as estrous cycle, fertilization, embryo development, and maternal environment are of paramount importance. However, fundamental information on reproductive and productive efficiency, surrounding environment, functional genome information, and their interrelationship are also equally important. Hence, precise and early selection of superior dairy animals based on the partial or whole genome, proteome data, and accurate association could be future choices, provided a large number of animals with accurate pedigree under a systematic farming system become available.

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McKay, S.D., Schnabel, R.D., Murdoch, B.M., Matukumalli, L.K., Aerts, J., Coppieters, W., Crews, D., Neto, E.D., Gill, C.A., Gao, C., Mannen, H., Stothard, P., Wang, Z., Van Tassell, C.P., Williams, J.L., Taylor, J.F., Moor, S.S., 2007. Whole genome linkage disequilibrium maps in cattle. BMC Genet. 25 (8), 74. Meuwissen, T., Hayes, B., Goddard, M., 2001. Accelerating improvement of livestock with genomic selection. Annu. Rev. Anim. Biosci. 1, 221e237. Misztal, I., Legarra, A., Aguilar, I., 2009. Computing procedures for genetic evaluation including phenotypic, full pedigree, and genomic information. J. Dairy Sci. 92, 4648e4655. Mout, R., Ray, M., Lee, Y.W., Scaletti, F., Rotello, V.M., 2017. In vivo delivery of CRISPR/Cas9 for therapeutic gene editing: progress and challenges. Bioconjugate Chem. 28, 880e884. Mrode, R., Aliloo, H., Strucken, E.M., Coffey, M., Ojango, J., Mujibi, D., 2018. The impact of modelling and pooled data on the accuracy of genomic prediction in small holder dairy data. In: Proceedings of the World Congress Applied to Livestock Production (Auckland). Neves, H.H., Carvalheiro, R., Brien, R.A.M.O., Utsunomiya, Y.T., do Carmo, A.S., Schenkel, F.S., 2014. Accuracy of genomic predictions in Bos indicus (Nellore) cattle. Genet. Sel. Evol. 46, 17. O’Connell, M.R., Oakes, B.L., Sternberg, S.H., East Seletsky, A., Kaplan, M., Doudna, J.A., 2014. Programmable RNA recognition and cleavage by CRISPR/Cas9. Nature 516, 263e266. Pryce, J.E., Daetwyler, H.D., 2012. Designing dairy cattle breeding schemes under genomic selection: a review of international research. Anim. Prod. Sci. 52 (3), 107e114. Richard Pursley, J., Silcox, R.W., Wiltbank, M.C., 1998. Effect of time of artificial insemination on pregnancy rates , pregnancy loss and gender ratio after synchronization of ovulation. J. Dairy Sci. 81 (8), 2139e2144. Schmitt, A.O., Jens, A., Bortfeldt, R.H., Brockmann, G.A., 2010. CandiSNPer: a web tool for the identification of candidate SNPs for causal variants. Bioinformatics 26 (7), 969e970. Sellner, E.M., Kim, J.W., McClure, M.C., Taylor, K.H., Schnabel, R.D., Taylor, J.F., 2007. Board-Invited Review: applications of genomic information in livestock. J. Anim. Sci. 85 (12). Silva, R.M.O., Fragomeni, B.O., Lourenco, D.A.L., Magalhães, A.F.B., Irano, N., Carvalheiro, R., 2016. Accuracies of genomic prediction of feed efficiency traits using different prediction and validation methods in an experimental Nellore cattle population. J. Anim. Sci. 94, 3613e3623. Sonesson, A.K., Meuwissen, T.H.E., 2009. Testing strategies for genomic selection in aquaculture breeding programs. Gent. Sel. Evol. 41, 37. Sorensen, A.C., Sorensen, M.K., 2009. Inbreeding rates in breeding programs with difference strategies for using genomic selection. No.40. In: Proceedings of the 2009 Interbull meeting. Strucken, E.M., Al-Mamun, H.A., Esquivelzeta Rabell, C., Gondro, C., Mwai, O.A., Gibson, J.P., 2017. Genetic tests for estimating dairy breed proportion and parentage assignment in East African crossbred cattle. Genetics. Sel. Evol. 49, 67. Su, G., Guldbrandtsen, B., Gregersen, V.R., Lund, M.S., 2010. Preliminary investigation on reliability of genomic estimated breeding values in the Danish Holstein population. J. Dairy Sci. 93. Terakado, A.P.N., Piccoli, M.L., Carvalheiro, R., Schenkel, F.S., Fonseca, L.F.S., 2014. Albuquerque L.G., Accuracy of genomic selection for growth traits in Nellore cattle. In: Proceedings of the 10th World Congress of Genetics Applied to Livestock Production, Vancouver, BC. Van Raden, P.M., 2008. Efficient methods to compute genomic predictions. J. Dairy Sci. 91, 4414e4423. VanRaden, P.M., Van Tassell, C.P., Wiggans, G.R., Sonstegard, T.S., Schnabel, R.D., Taylor, J.F., Schenkel, F.S., 2009. Invited review: reliability of genomic predictions for North American Holstein bulls. J. Dairy Sci. 92, 16e24. Veerkamp, R.F., Beerda, B., 2007. Genetics and genomics to improve in high producing dairy cows. Theriogenology 68 (Suppl. 1), S266eS273. Wangkum hang, P., Wilantho, A., Shaw, P.J., Flori, L., Moazami-Goudarzi, K., Gautier, M., 2015. Genetic analysis of Thai cattle reveals a South east Asian indicine ancestry. PeerJ 3, e1318. Washburn, S.P., White, S.L., Green, J.T., Benson, G.A., 2002. Reproduction, mastitis, and body condition of seasonally calved Holstein and Jersey cows in confinement or pasture systems. J. Dairy Sci. 85, 105e111. Weerasinghe, W.M.S.P., 2014. The accuracy and bias of estimates of breed composition and inference about genetic structure using high density SNP markers. Ph.D. Thesis. In: Australian Sheep Breeds. The University of New England. Werner, F.A., Durstewitz, G., Habermann, F.A., Thaller, G., Kramer, W., Kollers, S., 2004. Detection and characterization of SNPs useful for identity control and parentage testing in major European dairy breeds. Anim. Genet. 35, 44e49. Zumbach, B., Jorjani, H., Durr, J., 2010. Brown Swiss genomic evaluation. Interbull Bull. 42, 44e51.

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Chapter 11

Gene therapy Deepti Saini Protein Design Pvt. Ltd, SID, Indian Institute of Science, Bangalore, Karnataka, India

11.1 Genes Genes, segments of DNA present within the chromosomes of a cell, provide the code for conversion RNA into proteins, the functional and structural units of the human body. Hence, a gene is a template. These proteins determine the properties of our cells and their function, including the immune system. Genes can be altered through genetic mixing from parent to offspring, aging, and mutation. Genes are transmitted from parent to offspring, each parent providing one half of the set of total genes. This is the reason that traits such as hair and eye color are linked to genes, but defects in the genes can be transferred to the offspring. These lead to genetic diseases. A genetic disease is caused due to abnormalities in genetic makeup. The genetic abnormality can be from a discrete change in a single base in the DNA of a gene to an overall chromosomal abnormality involving the addition or subtraction of a single chromosome or a set. Some genetic disorders are inherited, while acquired mutations in a gene or group of genes may cause others. Some genetically inherited disorders include single-gene inheritance, multifactorial inheritance, chromosome abnormalities, and mitochondrial inheritance. Single-gene inheritance, also called mendelian or monogenetic inheritance, is caused by mutations that occur in the DNA sequence of a single gene. Single-gene disorders show inheritance in different patterns: autosomal dominant inheritance, where only one copy of a defective gene can cause the disease; autosomal recessive inheritance, where two copies of a defective gene are mandatory to cause the condition; and X-linked inheritance, in which the defective gene is present on the X-chromosome. Some examples include cystic fibrosis, thalassemia, sickle cell anemia, Huntington’s disease, and hemochromatosis. Multifactorial or polygenic inheritance is associated with heritable traits such as fingerprint patterns, height, and eye or skin color. A combination of environmental factors and mutations in multiple genes give rise to these disorders. Different genes that influence breast cancer susceptibility have been located on chromosomes 6, 11, 13, 14, 15, 17, and 22. A few common multifactorial disorders include heart disease, high blood pressure, Alzheimer’s disease, cancer, and obesity.

11.2 Gene therapy Mutations, insertions, deletions, and alterations in genes or its regulatory elements may result in reduced/absent production of encoded proteins or expression of structurally or functionally abnormal proteins, leading to genetic disorders. Gene Therapies (GTPs) work by repairing, replacing, or deactivating dysfunctional genes that cause disease. The complexities of GTPs, their design, and production, pose challenges for their translation into clinical applications. A gene can be naturally altered as it ages, by many factors such as ionizing radiation, chemical carcinogens, or errors in DNA replication. A commonly used method to treat these genetic errors is through the removal or alteration of the changed gene. The new or altered gene changes how the proteins are produced. A viable method to treat these genetic diseases involves the use of vectors to transfer healthy genes, as pharmaceuticals cannot always cure a malfunction in the genetics of a person. Researchers are trying to use gene-editing techniques such as CRISPR to precisely change problematic DNA sequences or even genetically modifying immune cells to enhance their cancer combating ability. Recently, a variety of treatments, where disease-carrying genes are replaced in their entirety by foreign DNA, are developed. The foreign genetic material is functional DNA, which is synthesized in the laboratory. This DNA is to be inserted into the genes of the patient, via a vector, since nuclear material is not taken up by the nucleus of the cell by itself. Viral vectors

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vary in the extent of transferring genes to the cells that they are able to recognize and are able to infect, and whether they are capable of altering the cell DNA permanently. Viral vectors are modified to beneficially alter the host gene rather than harmfully. Viruses are best for this function as they have evolved efficient mechanisms of transferring genetic information to the host cell. Utilizing harmless viruses such as adeno-associated viruses (AAVs) can reduce the chances of a negative reaction. Nonviral vectors have a lower success rate (Electroporation, direct DNA injection). The viruses’ gene-altering capabilities are utilized to insert useful and functional, healthy genes. The types of viruses used for Gene Therapy are typically nonpathogenic, or the disease-causing genes are removed. The vectors are extensively tested before actual use to ensure the safety of the patient. These vectors can be inserted outside the body in ex-vivo treatment and inside the body in in-vivo treatment. This is done through injection or IV, rather than by surgical methods or medication. The most commonly utilized viruses AAVs are typically small and not known to cause diseases. This severely reduces the chances of a negative reaction and adverse side effects. Inherited single-gene defects can now be treated and cured with the patient’s own bone marrow stem cells that have been engineered with a viral vector containing the missing gene. Local or systemic injection of viral vectors has been used to treat patients with inherited retinopathies and hemophilia B. There is also promise in the areas of cancer and infectious disease. The future holds improvements in the safety, efficacy, and manufacture of gene delivery vectors. Recently, bacterial vectors have emerged as a promising tool to deliver very large DNA cargo, such as chromosomes, but knowledge about their potential applications and pitfalls are still emerging. Other nonviral methods such as naked DNA transfection using chemicals polymers or biolistic methods like nanoparticle, liposome, dendrimer, etc., mediated delivery of nucleic acid sequences typically elicit a less immune response but have lower efficacy than viral vectors. Advances to overcome these hurdles: Creation of extensively gene-deleted less immunogenic vectors, have reduced insertional mutagenesis potential, improvement of the GTP efficiency of the ex-vivo transduction, increasing the tissue specificity, efficiency of in-vivo gene transfer and transgene expression by using tissue-specific and/or inducible promoters, expanding the vector types for enhanced tropism and avoiding preexisting immune response by developing alternative viral serotypes, and identification of new viral species or new materials/nanomaterials for use as vectors,and in addition to recombinant viral vectors, conditionally replicating viruses (Oncolytic virus), which show promise in tumor-directed gene therapy. Nonviral vector systems that have applications for gene delivery include nanoparticles or nanocomposites. Such vectors can be designed to incorporate carbohydrate/glycopeptide motifs for targeted receptor-mediated delivery to specific cell types in vivo. There are also studies being done at the University of Wisconsin for discovering viral vectors alternatives. These offer hope for new treatments for inherited diseases, some cancers, and even certain difficult viral infections. The usual method for delivering genes to the specific tissues in the body has the risk of complications. They have addressed some of the problems of viral vectors by packing a gene-altering payload into a tiny, customizable, synthetic nanocapsule. They have described the delivery system in the journal Nature Nanotechnology in 2019.

11.3 Use of gene therapy Gene Therapy is used to treat genetic diseases that are extremely harmful, rare, and life-threatening. The definition of a rare disease is dependent on the number of individuals affected by a disease per total number in a group. A rare disease is one that affects between one in 1000 and one in 200,000 people. There are between 5000 and 8000 rare diseases in the world, with most having a genetic element to them (Field and Boat, 2010). GTP is a promising treatment for treating conditions such as cystic fibrosis and hemophilia, strongly linked to genes. It is also a promising method for remedying cancer, which is not an inheritable disease, but involves gene mutations. Cancer is caused by damage to genes, which, in turn, causes abnormal cell growth or uncontrollable cell division. Using GTP to alter these faulty genes may be able to stop the spread of the disease. Clinical trials have been done to mediate diseases such as Sickle Cell Anemia, Cystic Fibrosis, Hemophilia, Huntington’s disease, Parkinson’s disease, Alzheimer’s Disease, etc. The most notable diseases include Severe Combined Immune Deficiency (SCID), Hemophilia, and Chronic Granulomatous disorder (Ginn et al., 2018). Adenosine Deaminase (ADA) Deficiency related SCID is a severe condition, which involves a deficiency of the enzyme ADA as the body is unable to synthesize it. This leads to a severely compromised immune system, and the affected patients succumb to severe infections without bone marrow transplants. Because of this, the patients have to stay within bubbles. But a therapeutic gene (ADA gene), was introduced into the bone marrow of the patients (ex-vivo) and then transplanted back into them. The immune system was made functional, with little to no side effects. The treated individuals now lead normal lives (Mavilio and Ferrari, 2008).

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Hemophilia is a rare condition in which patients are unable to form blood clots. This is due to a lack of blood clotting proteins, which are mediated by genes. This can cause severe bleeding in the case of large cuts or excessive bruising due to internal bleeding. It is a genetic condition linked to the X-chromosome. Gene Therapy is used as a treatment for this condition as it can alter the dysfunctional genes in the liver cells to begin producing clotting factors. But this treatment is transient as the altered cells are usually rejected by the patient as foreign. The outcome might be more successful if immunosuppressant drugs are utilized, along with Gene Therapy (Gollomp et al., 2019). Chronic Granulomatous disorder is a genetic condition where the immune system malfunctions. Patients suffering from this disease usually have chronic and recurrent infections. This causes locations of inflammation in various tissues that damage them and can be fatal. Using similar techniques to the ADA-SCID cases, successful transplantation of marrow resulted in functional immune systems for two patients (Segal et al., 2011). For inherited disorders with a dominant defective gene, such as Huntington’s disease, gene therapy provides the opportunity to change the expression of the pathogenic gene in the tissues with the worst symptoms. There is also the possibility of repairing genes in recessive genetic disorders. Initial strategies for these technologies, synthetic combinations of zinc finger DNA-binding domains are used to target a mutated genomic sequence, coupled to an endonuclease domain to create a zinc finger nuclease (ZFN). When expressed in human cells, this enzyme induced a double-stranded break in the genomic target, which nullifies the gene’s transcription and translation. This is then repaired by homologous recombination with a correct template supplied (Collins and Thrasher, 2015). Homing endonucleases or mega-nucleases are enzymes that cut genomic DNA within the cells that synthesize them at a very low frequency at specific sites. As the host cell repairs these specific sites, this results in copying of the homing endonuclease gene into the cleavage site, hence the usage of the term “homing.” I-SceI mega-nuclease has been designed to target specific sites in the mammalian genome (Wang et al., 2014). Transcription activator-like effectors (TALEs), transcriptional activators from Xanthomonas, are comprised of a string of about 30 amino acids, each of which binds to a single target base in a DNA sequence. Engineered TALE-nuclease chimeras (TALENs) can also be used for genome cleavage at targeted sites and in mammalian cell repair (Sanjana et al., 2012). As these methods for editing the cellular genome have been adapted, this has also allowed targeted integration of the expression cassettes to “safe harbors” such as the AAVS1 site on chromosome 19 or the endogenous locus. For achieving this, the expression cassette must be flanked with DNA surrounding the nuclease cleavage site to direct homologous repair and also be delivered efficiently, perhaps with a nonintegrating lentiviral vector (Lee et al., 2019; Philpott and Thrasher 2007; Cockrell and Kafri, 2007). There is also research being done to utilize gene therapy to treat cancer, mesothelioma, and neurodegenerative diseases such as Parkinson’s disease. Gene therapy can also be used to improve the effectiveness of existing drugs, improving the ability of body cells to be treated with other drugs. Temozolomide (TMZ), a drug used to treat glioblastoma, a type of brain cancer that causes astrocytes to accumulate in the brain. TMZ is usually used in combination with O6-benzylguanine (O6BG) to inhibit methylguanine methyltransferase (MGMT) enzyme. MGMT enzyme is overexpressed by many glioblastomas and inactivates TMZ. The amount of O6BG that can be used is limited, as it is very toxic to hematopoietic cells. Gene therapy has been utilized to express the O6BG-resistant MGMT mutant gene in hematopoietic cells of glioblastoma patients, allowing them to receive more intensive chemotherapy (Jiapaer et al., 2018).

11.4 Types of gene therapy: somatic and germline Technically, all cells in the human body contain genes and can be potential targets for gene therapy. However, they are divided into two major categories: somatic cells (most cells of the body) or cells of the germline (eggs or sperm). Somatic Gene Therapy, in which genes are transferred into somatic cells, and Germline Gene Therapy, in which genes are inserted into germ cells. Somatic therapy is used as a treatment for individuals. Germline involves the transfer of genes into gametes and germ cells, thereby ensuring that all the alterations are present in inheritable condition, and used to treat further generations also. If the transfer of gene is done early during embryologic development, the gene transfer could happen in all cells of the developing embryo. Germline gene therapy is appealing due to its potential for offering a permanent therapeutic change for all those who will inherit the target gene and eliminating disorders from a particular family, and eventually from the population, forever. Many clinical trials are currently utilizing SCGT to treat immunodeficiency, hemophilia, cystic fibrosis, etc. This is only a partial treatment as a complete replacement of multiple genes is currently not possible. It is a safer approach as it affects only the targeted cells. But the effects of this type of therapy are short-lived, as most cells of tissues are gradually replaced, and hence, repeated treatments are required. All gene therapy human trials have been Somatic, as Germline Therapy has ethical complications. Somatic Therapy has two broad subdivisions, ex-vivo, and in-vivo.

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In Germline Gene Therapy (GGT), germ cells are modified by introducing different or functional genes into their genome. Many countries around the world do not allow Germline Therapy due to ethical and technical reasons. There may be possible risks to future generations, and there is currently insufficient knowledge about this type of therapy to utilize it successfully. This therapy is also controversial, as it has been compared to “Playing God,” where humans can control the traits that are to be passed onto their offspring. Gene alteration may be a more convenient, reliable, and cost-effective method of supplying biological medicines required systemically. The first licensed gene therapy medicine alipogene tiparvovec (Glybera) is an AAV1 vector carrying human lipoprotein lipase (LPL), injected intramuscularly for the treatment of patients with LPL deficiency. When the vector carrying the LPL is injected, the enzyme is released by the vector and counters the enzyme deficiency. Recently, there has been increased interest in using antibody gene delivery systems to muscle cells for the treatment of infectious diseases. In HIV, permanent expression of a broadly neutralizing antibody could provide more effective protection than vaccination, which has thus far failed, as viruses are constantly adapting. The effectiveness has been demonstrated in a humanized mouse model (Collins and Thrasher, 2015). This technology has also been tested in mice for influenza prophylaxis, delivered to muscle or intranasally (Kang et al., 2012). Antibody gene therapy as prophylaxis also has the capability to be very effective in a rapidly spreading pandemic, where vaccination might be too slow. In this type of application, a clinically compatible small molecule that could regulate gene expression in vivo would be very useful. The antibiotic selection systems used in vitro (such as the Tet-on system (Das et al., 2016)) are unsuitable in vivo because the bacterial tetracycline-controlled trans-activator is immunogenic, leading to transduced cell elimination. Due to gene delivery, the generation of active drugs at the site where they are needed is possible. Parkinson’s disease is caused by a deficiency of dopamine in the brain. The dopaminergic neurons, die leading to loss of movement control and tremors. The common treatment for Parkinson’s disease is a tablet delivering a dopamine precursor combined with a drug to enhance bloodebrain barrier permeability of dopamine. This works well in early-stage diseases but declines in effectiveness as time progresses. A gene therapy approach, directly delivering the enzymes to produce dopamine transferred into the brain, might provide more stable local dopamine concentration. The first clinical trial used a lentiviral vector and was reported as safe and had some level of effectiveness, but more efficient delivery or higher gene expression will be necessary for furthering this method.

11.5 Types of vectors The organisms and methods by which transfer of the genetic material during Gene Therapy occurs are called vectors. Viral vectors introduce the genetic material into the host cell by joining it to the cell chromosomes, which use them as a blueprint for the manufacturing of proteins. Different types of viruses have been utilized for gene therapy, such as Retroviruses, Adenoviruses (McConnell and Imperiale, 2004), and AAVs (Wu et al., 2006; Schultz and Chamberlain, 2008), herpes virus (Oehmig et.al., 2004), etc. Nonviral methods of Gene Therapy are complex and involve direct transfer of the genes into the host. These include the gene gun, electroporation, sonoporation, injection of naked DNA, etc. They have several advantages over viral vectors, but also some disadvantages. Some advantages include the ability to have large scale production and the lack of risk factor associated with viruses, but the disadvantage of nonviral methods is that there is reduced levels of DNA integration and expression of the new genes, lowering overall effectiveness.

11.6 Techniques of gene therapy Gene augmentation, replacement of a mutated copy of the gene by a healthy copy (via recombination), correction of mutation by gene editing (CRISPR, TALEN, ZFN, etc.) methods, silencing of a dominant mutation (via shRNA or gene editing), altering the expression of genes by affecting transcription (transcription factors, epigenetic modulators) or splicing (exon skipping) are all considered to be GTP. Techniques of Gene Therapy include Gene augmentation, inhibition, and specific cell targeting. Gene Augmentation Therapy, when cells contain nonfunctional DNA, which prevents the formation of final protein, functional DNA is inserted. Gene Inhibition Therapy helps treat disorders such as cystic fibrosis; if there is a faulty gene in the cell, a gene is inserted, which either suppresses or overrides the faulty gene. This is used to treat cancer and inherited diseases and Specific Cell Therapy, where gene alteration is done for specific faulty cells in cancer treatment. It can be done by the insertion of a suicide gene to kill the cell or a marker gene to allow the cell to be targeted by the immune system. Gene Augmentation involves altering the original nonfunctional gene and inserting a functional gene in its place. A new, functional gene is inserted in the place of the mutated code to correct the translation of the gene into proteins.

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The nonfunctional gene begins to function correctly. This can only be used to treat conditions where the effects of the disease are curable, and the body is not damaged enough to be unable to recover. Gene augmentation can treat functional disorders such as cystic fibrosis. Gene Inhibition involves altering a malfunctioning gene by replacing the faulty gene with a blocking gene, preventing the improper function. The faulty gene produces products that are unhealthy for the individual. This can be used to treat infectious diseases caused by viral tampering and cancer, as well as inherited conditions. This type of therapy aims to produce a final gene that either suppresses the expression or interferes with the production activity of the gene. This is very useful to treat only specific cells such as in cancer, where oncogenes, genes that promote cell growth and division, are malfunctioning. Oncogenes, when overactivated, lead to excessive cell division and metastasis, causing cancer. If these genes are inhibited, then the cancer cells begin to stop dividing. Specific Cell Targeting is done to treat diseases such as cancer, which involve groups of cells that are faulty and disease-causing. In this method, DNA is inserted into specific disease-causing cells to cause cell death. There are two methods of Specific Cell Targeting, insertion of suicide genes, and marker genes. Suicide genes are inserted into the target site, which integrates with the specific cell gene. These genes, when translated, produce highly toxic products, which cause cell death. Marker genes, when inserted into the target site, integrate with the specific cell genes which are present on the surface of the specific cells. These behave similarly to antigens, which are chemicals recognized by our body’s immune system antibodies. This leads to the body recognizing the cells as pathogens, which leads to the immune system having a natural response and killing the foreign cells. The most successful method of gene delivery involves synthetic particles, using lipids or polymers to carry DNA. However, these methods have not yet achieved efficient uptake and sustained gene expression in vivo. Retroviruses integrate their genome into host cell DNA, and when the infected cell divides, the integrated provirus is transmitted to daughter cells. A murine leukemia virus (MLV) was the first retroviral genome, which was engineered to carry a foreign gene, HSV thymidine kinase (Tabin et al., 1982; Nienhuis et al., 2006; Berges et al., 2007). Deletion of the sequence which codes for packaging the viral RNA into particles allowed viral genes required for particle production to be provided in cis, which means they were transferred into the regions of noncoding DNA, which regulates the transcription of neighboring genes. These were the replication-defective MLV vectors carrying no viral genes (Logg et al., 2001). Viral packaging cell lines were then constructed with the viral genes expressed from two segments of DNA, and this eliminated the risk of recombination with the vector, which may generate a replication-competent virus. The ability of these MLV vectors to deliver genes to mouse and human bone marrow stem cells was demonstrated. This was because a number of inherited diseases could be cured by bone marrow transplantation from a suitable donor. Therefore, gene therapy using the patient’s own cells engineered to carry a correct copy of the faulty coding sequence was an attractive option for patients without a fitting donor. The feasibility of this gene transfer to patients was first shown by Rosenberg et al. (1990) by utilizing an MLV vector to introduce the gene for resistance to neomycin into tumor-infiltrating lymphocytes before infusing the cells into five recipients with advanced melanoma.

11.7 History of human gene therapy In the early days of the gene therapy research, more than 2 decades ago, gene therapy was similar to many experimental medicine approaches and was hindered by the occurrence of rare but severe side effects. But after extensive research, there has been the development of highly sophisticated gene transfer tools with improved safety and efficiency. Additionally, highly specific and site-directed gene-editing technologies have been developed and applied clinically. Gene Therapy, as a concept, was first proposed by Stanfield Rogers and Peter Pfuderer in 1968. They proved that transfer of foreign genetic material is possible using viruses, using the Tobacco Mosaic Virus to introduce a poly-adenylate stretch to the viral RNA. In 1984, there was a retrovirus vector system that was designed to successfully insert foreign genes into mammalian chromosomes initiating the trials of human gene therapy testing. The first approved trial for gene therapy took place in 1990, where Ashanti DeSilva, a 4-year-old suffering from ADASCID, was given treatment for the condition, which caused severe immunodeficiency at the National Institutes of Health (NIH), under his direction. The method used was the Gene Augmentation technique to replace the dysfunctional gene by the functional gene. This helped the patient to begin production of the deficient enzyme. Unfortunately, the functional cells did not replicate to produce functional daughter cells. Because of this, the effects were temporary. Following this, cancer gene therapy trials began in 1992. The first treated cancer was a fatal brain tumor, glioblastoma multiforme. The trial was done utilizing a vector expressing the antisense IGF-I RNA, which was effective due to the antitumor mechanism of the IGF-I antisense. This gene is related to apoptotic phenomena. Hereditary diseases were first treated in 1992 by Claudio Bordignon, at the Vita-Salute San Raffaele University. He performed the therapy by using

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hematopoietic stem cells as vectors. The first treatment for ADA related SCID was done in 1993, for the patient Andrew Gobea. The prenatal screening revealed the condition, and stem cells for the treatment were taken from the placenta and umbilical cord. Genes that code for the enzyme for ADA were inserted into the stem cell DNA. This caused the WBCs in Andrew’s body to begin producing the enzyme using the functional gene. Cancer gene therapy was registered in 2002 by the Wiley gene therapy, trials no 635 and 636, which utilized the strategy of antisense IGF-I RNA using antisense/triple helix IGF-I. This method of therapy has helped in the treatment of cancers of the liver, prostate, uterus, ovary, and colon, as well as glioblastomas. This is an efficient method to prevent further spread of cancer, as it simultaneously stops the transcription and translation of the IGF-I gene and therefore prevents expression, as well as strengthening the apoptotic phenomena. In 2003, genetic material was successfully inserted into brain. Scientists utilized liposomes coated in polyethylene glycol, which can cross the bloodebrain barrier. Adenoviruses were also engineered to deliver the tumor-suppressor gene p53 for the treatment of squamous cell carcinoma in the head and neck. The chronic granulomatous disease was first treated in 2006 successfully. New methods were discovered to prevent the rejection of genes. The immune system usually recognizes the new gene and rejects it as a foreign body, but this method utilizes microRNA to obscure the gene and prevent the natural immune response. Testing was done on mice, which had the gene transplant with the microRNA sequence. Rejection did not occur, so this was taken as a viable method to prevent immune response (Malech and Hickstein, 2007). Lentiviral vectors were first utilized in November 2006 to treat HIV infection. Five subjects with HIV infection were given an intravenous injection of CD4 T cells which had been altered with VRX496, which develops an antisense response against the HIV envelope. All five patients posttreatment had an increased immune response to the HIV antigens. From 2007 to 2009, gene therapies were done to treat conditions such as Lebers congenital amaurosis and adrenoleukodystrophy. Amaurosis is disease-causing blindness, due to a mutation in the RPE65 gene, was treated with recombined AAV carrying the functional gene. This was successful in treating the condition in three clinical trials in 2008. A single dose of Luxturna restored functional vision in 41 patients. In September 2010, there was a successful case of a patient in France suffering from beta-thalassemia. This rare inherited condition is responsible for the missing beta-hemoglobin in the patient’s body. The treatment involves using lenticel vectors to induce beta-hemoglobin production in the pureblood and bone marrow cells. The patient’s body contained only a third of the hemoglobin induced by the viral vector and this produced a normal healthy amount of hemoglobin. Cancer immunotherapy was also done in LA Sabana University in Bogota, where a modified antigene was used to target IGF-I expressing tumors. In 2011, the treatment of HIV was done by hematopoietic stem cell transplantation. This needed complete ablation of the bone marrow system, so it is a very difficult process. In the same year, chronic lymphocytic leukemia was treated and cured, using modified T cells, which removed the cells expressing the proteins causing the disease. In Russia, the first drug for the treatment of peripheral artery disease was registered, delivering the gene encoding VEGF, called Neovasculgen, and used CMV promoter. In 2012, the treatment for lipoprotein lipase deficiency was done using Alipogene tiparvovec, to prevent severe pancreatitis. This was the first time that the European Medicines Agency recommended gene therapy as treatment commercially. Alipogene tiparvovec was released at 1.6 million USD per treatment, the most expensive treatment in the world at the time. In March 2013, three out of five patients suffering from Acute Lymphocytic leukemia had been in remission after being treated with modified T cells to attack the cancerous B-cells. In April, the second phase of clinical trials began to combat heart disease. These trials were called CUPID2 and SERCA-LVAD, done to improve the muscle function in heart tissue by increasing the levels of SERCA2, a protein, which helped the muscles function efficiently. Till 2014, 18 children overall had been cured of ADA-SCID. January 2014 had patients treated for choroideremia, a genetic eye disease causing loss of sight, with AAV. By 2016, 32 patients had successful results. In March, research was conducted on HIV patients with a rare mutation (CCR5 deficiency) with promising results. In 2015, a treatment for beta-thalassemia called LentiGlobin BB305 led to many patients going off frequent blood transfusions usually needed to fight the disease. In March, a study for producing antibodies for malaria, influenza, and Ebola, was done by a technique known as immunoprophylaxis. A worldwide moratorium was called for, to prevent germline gene modification in humans until the ethical and scientific implications were fully understood. In April 2016, the second treatment to be approved in Europe was called Strimvelis, which treated ADA Deficiency. Other trials were also conducted for lung cancer treatment and cystic fibrosis. 2017 led to the treatment of nonHodgkin lymphoma, sickle-cell disease, and acute lymphoblastic leukemia. A form of adoptive cell transfer therapy for ALL was

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approved in the United States, which utilized engineered T-cells producing a chimeric T-cell receptor that causes the T cell to target the protein CD19 on B cells. In December 2017, Luxturna was approved by the FDA. This was the first in-vivo gene therapy, for the treatment of Leber’s congenital amaurosis. 2017 also saw two gene therapy drugs approved for the US market,: Yescarta (Gilead) and Kymriah (Novartis). CAR-T involves taking blood from a patient, genetically reengineering specific blood cells, and finally infusing these modified cells back into the sick patient’s body. In CAR-T therapies, the patient’s T-cells are injected with a new synthetic gene (CAR). The CAR gene translates into a receptor protein that sits on the outside of the T-cell and helps the T-cell attack specific types of cancer. In 2017, the FDA approved the use of CAR-T therapy (Novartis designed Kymriah) for the treatment of ALL (acute lymphoblastic leukemia). The T-cells are genetically tailored to target a marker (CD19) present on cancerous B-cells. B-cells are overproduced in ALL patients. In October of 2017, the use of another CAR-T therapy (Yescarta) developed by Kite Pharma to treat adults with diffuse large B-cell lymphoma, the most common form of NHL (nonHodgkin lymphoma) was approved by the FDA. Gene therapies such as Glybera, Stremelis and Gendicine have already been approved in Europe and China. While CAR T therapy induces a high complete response rate in B-cell acute lymphoblastic leukemia, or B-ALL, Majzner and Mackall found in their analysis that long-term follow-up assessment of patients has shown high incidences of relapse limiting overall success. CAR T-cell therapy (CAR-T) was pioneered by scientists Isabelle Rivière, Michel Sadelain, and Renier Brentjens at Memorial Sloan Kettering Cancer Center. The team has had many breakthrough discoveries, which have led to clinical studies in the United States and worldwide involving pediatric and adult patients. The studies done on children and adults, by Majzner and Mackall, have shown a lot of remission rates in clinical trials without requiring additional treatment. However, cost remains a concern for Kymriah and Yescarta. Both treatments are quite expensive. Kymriah costs about $475,000 for a single infusion, whereas Yescarta costs about $373,000. There has also been a CAR-T case study recently done that has shown promise in treating acute myeloid leukemia done by H. Lee Moffitt at the Cancer Center and Research Institute. Chimeric Antigen Receptor T-cell therapy, also known as CAR-T therapy, was one of the biggest research breakthroughs of 2017 by the American Society of Clinical Oncology. This form of gene therapy utilizes the patient’s immune cells to fight cancer by making the cancer cells visible to the immune system. The FDA has approved CAR-T Therapy for adults suffering from diffuse large B-cell lymphoma, as well as in young adults suffering from acute lymphoblastic leukemia. In the CAR-T treatment, T cells are removed from the patient’s blood and genetically modified in the laboratory to enable them to be able to identify and attack cancer cells. After the new cells are received, the patient receives preconditioning chemotherapy. This depletes their current immune system and allows room for the new cells. In the THINK study, preconditioning chemotherapy was not necessary. This case shows that CAR-T therapy is a viable option for patients, according to Sallman. In 2019, there had been a major step forward in gene therapy due to a study done in February by scientists operating with Sangamo Therapeutics. They showed the first-ever in-vivo gene therapy that decidedly modified the patient’s genes. The patient was suffering from Hunter Syndrome, and permanent alteration has been done. In May 2019, treatment for spinal muscular atrophy had been done by Zolgensma, an FDA-approved drug costing 2.125 Million USD, the most expensive drug ever to exist. By using an AAV9 viral vector, this gene therapy delivers SMN protein into the motor neurons of affected patients. Zolgensma is injected into the patient’s blood intravenously, and then the AAV9 virus can target neurons and deliver the SMN gene into the DNA in these neurons. It has been only 25 years since the feasibility of gene transfer to patients was first demonstrated (Rosenberg et al., 1990). So in the last 25 years, considerable and constant progress has been made, and a number of gene therapy applications have been discovered, providing benefits to patients in clinical trials. Proteins such as Antibodies are likely candidates for this method of gene delivery, to prevent infectious disease or to provide rapid prophylaxis, and also to treat cancer or autoimmune disease. Other familiar biological medicines like insulin may be replaced by gene therapy. Many inherited monogenic disorders treated by bone marrow transplantation with virally modified cells may be open to gene correction. To overcome this ineffectiveness, it will likely remain a delivery challenge. This is because there is not yet a simple way to deliver genes to a significant proportion of cells in tissues such as the lung epithelium or skeletal muscle. Commercial gene therapy activities are more likely to focus in the long term on gene addition therapy for common diseases such as heart disease or cancer, rather than temporary cures for muscular dystrophies. Injectable vectors are also more attractive in the form of licensed medicines because then they can be manufactured and distributed in a conventional manner like licensed medicines. For treatments requiring ex-vivo cell modification, there is a continuous requirement for local production of gene-modified cells. Thus, more efficient production and purification methods for viral vectors are required. Current protocols operate based on scaling up of laboratory methods, which just multiplies the cost proportionally.

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Among neurological diseases, severe genetic neurodegenerative conditions have been the focus of primary clinical applications. Complex neurodegenerative diseases, particularly Parkinson’s disease also have been utilizing Gene therapy for treatment, with promising results in human patients, which demonstrates that specific targeting of central nervous system (CNS) cells in the brain and spinal cord, is a viable method for treatment and highlights that access to the CNS using the viral vectors is achievable. On December 23, 2016, Spinraza (nusinersen), an antisense oligonucleotide, was approved by the US FDA to target spinal muscular atrophy patients. At the Association for Glycogen Storage Disease’s 41st Annual Conference, September 2019, Dr. David Weinstein of UConn School of Medicine presented his pioneering, 1-year clinical trial outcomes for the World’s First novel gene therapy treatment for glycogen storage disease (GSD). The rare and deadly genetic disorder of the liver, GSD type Ia, affects children from childhood through adulthood, causing frighteningly low blood sugar levels and constant need for glucose intake as cornstarch every few hours to survive. If a glucose dose is missed, the disease can lead to seizures and even death. Weinstein, whose team first administered the investigational gene therapy on July 24, 2018, at UConn John Dempsey Hospital in Connecticut, says the results are remarkable. The development of lung cancer treatment has come a long way within the last decade. The advancement of gene therapy through clinical trials till 2019 is contributing to millions of lives extended and more patients going into remission. An uncommon cancer affecting the lungs, mesothelioma, will hopefully start to leverage these types of treatments soon. The US Food and Drug Administration (FDA) has lifted a clinical hold and accepted an Investigational New Drug Application (IND) for an experimental sickle-cell disease treatment being codeveloped by Vertex Pharmaceuticals and CRISPR Therapeutics. Earlier, the FDA had placed a hold on the trial and IND for CTX001, an investigational geneediting treatment, making concerns out of questions that had not been addressed in the IND. Backed by a Harvard professor, a start-up is planning to reverse aging in dogs using gene therapy with an aim to achieve the objective of having the body and soul of a 22-year-old but the experience and skills of a 130-year-old. If the results show success, the same could be applied to humans. Rejuvenate Bio, a company cofounded by a pass out from Harvard Medical School, George Church, has already done time travel like tests on beagles and claims that it will make animals seem “younger” by inserting new DNA into their bodies. A clinical-stage gene therapy company, Arthrogen, together with the Center for Human DrugResearch (CHDR), a Leiden-based clinical research organization, proclaimed that a cohort of the first three patients with arthritis is undergoing an innovative phase Ib gene therapy testing for treatment of arthritis with the help of ART-I02. ART-I02 is an AAV5 vector encoding the IFN-b gene in humans under the control of an inflammation-responsive promoter. ART-I02 is designed to produce the antiinflammatory protein IFN-b in the synovial cells in joints. The goal is to achieve sustained clinical remission with a single treatment. This current clinical phase Ib trial evaluates the safety, tolerability, pharmacokinetics, immunogenicity, and antiinflammatory activity of ART-I02 treatment in patients with rheumatoid arthritis (RA) or osteoarthritis (OA). Totally, 12 patients with RA or OA in their wrists will be added. A similar phase Ib trial has begun in Canada, treating 15 patients with RA in their wrist joints.

11.8 CRISPR gene editing Alternative to gene therapy is genome editing. While Gene therapy delivers a new gene into cells to compensate for a Diseased or defective gene, genome editing removes or modifies the defective DNA in its original location within the genome. CRISPR gene editing is a method of altering the genomes of living organisms based on a simplified version of the bacterial antiviral defense system. CRISPR stands for Clustered Regularly Interspaced Short Palindromic Repeats, which are the main bacterial defense system that forms the basis for CRISPR-Cas9 genome editing technology. This method involves delivering the Cas9 nuclease along with a synthetic guide RNA into the cell, cutting the cell’s genome at the desired location, which allows the existing genes to be removed or altered or new ones to be added. Genomic editing in eukaryotic cells has been possible using various methods since the 1980s, but the methods used had been proven to be inefficient and impractical to implement on a larger scale. The Cas9 nuclease enzyme, working like genetic scissors, opens up both strands of the targeted sequence of DNA to introduce the modification by one of two methods. Knock-in mutations, which are facilitated through the Homology Directed Repair (HDR), is the traditional pathway of targeted genomic editing approaches. This allows for the introduction of targeted DNA damage and selective repair. HDR method requires the use of similar DNA sequences to drive the repair of the broken sequence through the incorporation of exogenous DNA to function as the repair template. This method works on the periodic and single occurrences of DNA damage at the target site to commence repair. Knock-out mutations caused by Cas9/CRISPR results in the repair of the double-strand break by means of NHEJ (NonHomologous End Joining). This End Joining can often result in random deletions or insertions at the repair site, disrupting or altering gene functionality. Therefore, genomic engineering by CRISPR-Cas9 allows researchers the ability to generate targeted random gene disruption and edit the genome.

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The predecessors to CRISPR gene editing were Zinc Finger Nucleases or ZFN’s. These are synthetic proteins whose DNA-binding domains enable them to create double-stranded breaks in DNA at specific points. They also act similar to scissors, just like the CRISPR gene editing method. In 2010, synthetic nucleases called transcription activator-like effector nucleases (TALENs) provided a simpler way to target a double-stranded break at a specific location on the DNA strand. Zinc Finger Nucleases, as well as TALENs, require a custom protein for each targeted DNA sequence to be created, which is a more difficult and time-intensive process than for guide RNAs since the DNA sequence has to be specific and doublestranded. CRISPRs are convenient to design because it requires making only a short RNA sequence. Compared to RNA interference (RNAi), which only partially suppresses gene function, CRISPR, ZFNs, and TALENs can result in full irreversible gene function knockout. CRISPR can also target several DNA sites simultaneously by simply introducing different guide RNAs. In addition, CRISPR costs are relatively low as compared to the other methods. The applications of CRISPR method are widespread and efficient, which help to generate transgenic models in the field of genetics. Cas9 is a special protein, which can play an important role in the immunological defense of certain bacteria against DNA-based viruses. This can easily be introduced into target cells via plasmid transfection along with guide RNA in order to model the spread of diseases and the cell’s reaction and defense mechanism in response to infection. The ability of Cas9 to be introduced in vivo permits the creation of more accurate models of gene function such as mutation effects, all while avoiding the off-target mutations observed with traditional methods of genetic engineering. The CRISPR and Cas9 revolution in genomic modeling does not only work to alter the genomes of mammals. The cells undergoing Cas9 treatment can be removed and reintroduced for boosting the effects of the therapy. CRISPR-Cas9 can be used to edit the DNA of organisms in vivo, and entire chromosomes can be modified in an organism at any point during its development. This method might be usefully employed for treating inherited aneuploid diseases as Down syndrome and intersex disorders, polyploid diseases caused by nondisjunction, or excessive chromosomes. CRISPR-Cas9 has been used to edit the genome in vivo of the basic organisms studied in most genetics, such as Escherichia coli, Saccharomyces cerevisiae, Candida albicans, Caenorhadbitis elegans, Arabidopsis and Mus musculus, etc. CRISPR can be utilized to create human cellular models of disease and their progress. For instance, applied to human pluripotent stem cells, CRISPR introduced targeted mutations in genes causing polycystic kidney disease (PKD) and focal segmental glomerulosclerosis (FSGS). These CRISPR-modified pluripotent stem cells were subsequently grown into human kidney organoids that exhibited disease-specific phenotypes. Kidney organoids from stem cells with PKD mutations formed large, translucent cyst structures from kidney tubules. Organoids with mutations in a gene linked to FSGS had junctional defects between podocytes, the filtering cells affected in that disease. This was because of the inability of podocytes to form microvilli between adjacent cells. Impressively, these disease phenotypes were absent in genetically identical background bearing control organoids, but lacking the CRISPR modifications. One of the clinical trials involving CRISPR, running in China, is testing the potential of the gene-editing tool to treat advanced esophageal cancer. The treatment being tested at the Hangzhou Cancer Hospital involves the extraction of immune T cells from the patient, and then the cells are modified to remove the gene that encodes for a protein called PD-1 using CRISPR. Some tumors are able to bind to this protein on the surface of the immune cell and instruct them not to attack. The modified cells are then reintroduced into the patient with a stronger ability to attack cancer cells. At least 86 people with different types of cancer have undergone treatment with CRISPR in China. In 2016, a healthy baby boy was born at a Mexican clinic. Having DNA from three parents, the boy had had his genes drastically engineered while still an embryo. Without this therapy, an inheritable neurological disorder would have killed him before the age of three. Researchers in China, in late 2018, under the Clinical project “Safety and validity evaluation of HIV immune gene CCR5 gene editing in human embryos” claim to have Genetically Engineered the First HIV-Immune Babies used genetic engineering tools (CRISPR) to create twins theoretically immune to HIV, smallpox, and cholera, MIT Technology Review has reported. This medical breakthrough is controversial, as many worry about eugenics and designer babies for the privileged. The twins, named Lulu and Nana, according to lead scientist He Jiankui of Shenzhen, are the result of in-vitro fertilization (IVF). A few weeks old, they appeared to be healthy. When they were at the single cell stage, genetic therapy using CRISPR removed the doorway through which HIV enters to infect people, to make them (theoretically) resistant to HIV infection. The edits did not hit just their intended target, and instead, the girls may be more prone to infections, have altered brain functions, and even experience earlier deaths causing a prickly debate on Germline Gene Therapy.

11.9 Gene therapy in animals Dr. Donald F. Patterson, founded the study of medical genetics in domestic animals, at the University of Pennsylvania. His vision recognized that human disease could be appreciated in animals if a deliberate search for them was undertaken. He worked with and also mentored numerous research veterinarians who carry out fascinating studies into the causes and cures

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of genetic diseases that afflict both humans and animals. John H. Wolfe wrote about Gene Therapy in Large Animal Models of Human Genetic Diseases. A major challenge to the medical sciences is the large number of disorders that are genetic in origin or involve a genetic susceptibility to disease-inducing environmental factors. These disorders include a wide variety of debilitating and fatal illnesses for which hardly any effective treatment or methods of prevention are available. Among these genetic abnormalities are congenital malformations, metabolic disorders, and cancer. Genetic variations also influence individual responses to pathogens and drugs. The structure and function of majority genes in animals are homologous to those in humans, and hence, many genetic diseases that occur in humans also occur in animals. Gene mutations also cause similar biochemical changes in functions, resulting in pathologic effects at the molecules, cell, organ, and organism levels. So, the knowledge gained through research in animals can be of direct benefit to human, as well as animal health. There have been extraordinary advances in the field of genetics in recent years. The techniques of molecular biology have made it possible to understand the underlying mechanisms of genetic disease at the level of DNA, RNA, and proteins. The successful sequencing of the human, mouse, dog, cat genome, and an expanding list of other species, as well as advances in genomics and related sciences, represent the onset of a new phase of insight making it possible to delineate the entire genetic program of individual types of cells and ultimately of whole tissues. In addition to that, these advances offer the prospect of making the right genetic defects and disease probabilities in animal and human patients through gene replacement therapy, manipulation of genetic pathways, engineering of stem cells, and delivery of recombinant proteins. The realization of practical benefits to both animal and human health will depend on a lot of future research. The increasing number and variety of transgenic, induced mutant, and naturally occurring animal models of genetic disease are vital for identifying new genes that cause disease, for understanding the cellular and molecular mechanisms of genetic diseases, and for elucidating the genes involved in diseases with complex inheritance patterns. A major use of such models is to help move gene therapy from proof of concept in cells and rodent models toward clinical trials in humans by allowing the investigation and solution of problems in scale-up and treatment of animal patients individually. The participation of veterinarians in these investigations is critical because they have the knowledge, experience, and skills in animal physiology, pathology, surgery, and medicine that enable them, with the proper scientific training, to effectively investigate vital aspects of the pathogenesis and treatment of gene-influenced diseases in animal homologs of human diseases. The studies focus on the use of large animal models of human genetic diseases for gene therapy research. The term “large animal” in this context refers to species other than the commonly used laboratory rodents. As is evident in the literature reviews, there are enough large animal models of human genetic diseases published (Horn et al., 2004; Casal and Haskins, 2006). The articles illustrate the variety of models available and have supported gene therapy research. The diseases cover diverse pathogenic mechanisms and affect various organ systems. Investigators are just beginning to explore the use of gene therapy methods to manipulate pathogenic processes in nongenetic diseases, as well as alternative therapeutic approaches such as stem cells, in large animal models of human disease, and the results will undoubtedly warrant further trials. The most useful classification of these trials can be by the organ system involved (eye, muscle, heart, serum, blood and immune cells, nervous system), which demarcate the diseases. However, quite a number of genetic diseases afflict multiple organs, and thus, there are reports of lysosomal (Haskins, 2009) and metabolic disorders (Koeberl et al., 2009) as well. The choice of organ system also depended, of course, on the availability of well-characterized models for investigation. A genetic disease can occur naturally in any species. A searchable database for animal Mendelian traits (OMIA) is maintained by The University of Sydney, similar to the Online Mendelian Inheritance in Man (OMIM), and the two databases can be cross-referenced. OMIA is accessible online directly (omia.angis.org.au) or through the National Center for Biotechnology Information (NCBI). Because some of the traits listed (e.g., coat color) are not diseases, there is a separate list of potential disease models, with 1029 such models in more than 25 mammalian species. Most models (w70%) occur in six species: dog, cat, horse, cow, pig, and sheep. Among these six, 31% of the diseases fall into just five categoriesdcongenital heart disease (63), lysosomal storage disease (Ellinwood et al., 2004; Haskins, 2009), dwarfism, inherited bleeding disorders, and inborn errors of metabolism (Bauer et al., 2009; Haskins, 2009; Sleeper et al., 2009). Other important groups of human diseases for which there are excellent large animal models are the muscular dystrophies and neurological disorders (Gagliardi and Bunnell, 2009; Wang et al., 2009). The suitability of each disease model for biomedical studies depends on the availability of both breeding stock to produce the disease and the amount of pathophysiological, biochemical, and genetic information about the model. For genetic disease models to become approved as laboratory animal models of disease, it is necessary to capture them in breeding colonies. Also, genetic diseases in these animals need medical diagnosis by veterinarians, followed by laboratory tests to confirm the genetic abnormalities. The diagnostic process is essentially the same as in human medicine as typically occurring in the pediatric population. An important point in using animal models to evaluate therapeutics for human translation is the comparability of the model to the human disease. Genetic diseases occur naturally in large animals and have been discovered by clinical

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ascertainment of the phenotype and its similarity to human disease. These disorders are thus true homologs of the human disease rather than modelsda homologous disease generally involves a mutation in an orthologous gene that causes similar biochemical, cellular, and organ abnormalities and results in clinical manifestations similar to human patients (Patterson et al., 1988). Mouse models are very useful for laboratory experimentation, but a number of induced genetic mutations in the mouse do not manifest the disease phenotype and thus are less useful for translational medicine investigations. Recently, researchers have produced a new range of highly desired disease models in large animals, a monkey model for Huntington’s disease (Yang et al., 2008) and a pig model to study cystic fibrosis (Rogers et al., 2008) by using transgenic and nuclear cloning technologies. These models should be especially useful for creating models of human diseases not naturally occurring in animals.

11.9.1 Large animal disease models The frequency of occurrence of individual genetic diseases in animal populations is rare, as in the human population. However, with animals, identification of at least one parent enables the establishment of a colony for further study. Mutations have been captured by breeding a single obligate carrier to a normal animal, then mating the F1 generation with the carrier parent (backcross breeding) until the mutation re-emerges. For many diseases, biochemical diagnosis can distinguish between a carrier and normal amounts of a protein (e.g., enzymatic activity levels) to assist in identifying the potential carriers. After identification of the gene and mutation, PCR assays enable tracking of the carriers of the mutation. There are a number of advantages to studying gene therapy approaches in breeding colonies of large animal models of human diseases. Body and organ sizes in large animals are similar to those of humans than rodent models. For blood disorders such as hemophilia, the blood volume is proportional to body weight; for example, a blood level of normal clotting factor from a transferred gene in a 40 kg dog closely estimates the therapeutic level for humans (Øvlisen et al., 2008). Similarly, a number of genetic diseases that affect the retina occur in dogs (Acland et al., 2001), and the architecture and large size of the canine eye provide excellent models both for the pathobiology and for surgical approaches. Researchers have developed clinical gene therapy trials for one form of retinal degeneration, as well as for hemophilia A and B directly from experimental studies in these dog models (Rawle and Lillicrap, 2004; Leber, 2008; Hasbrouck and High, 2008; Murphy and High, 2008; Nichols et al., 2009; Stieger et al., 2009). Testing experimental therapies under actual disorder conditions provide information about the effect on the diseased organs. In the brain, for example, genetic diseases typically manifest as lesions throughout the CNS, requiring global distribution of the gene vector or therapeutic protein. Brains in large animals provide a more accurate model of the conditions present in human neurodegenerative diseases (Vite et al., 2005) because (1) the cat and dog brain are 100 and 200 times, respectively, larger than a mouse brain, but only 10 to 30 times smaller than a human brain (Pierson and Wolfe, 2005); and (2) rodents have a smooth cortex versus the brains of larger animals that have a sulcated cortex and overall structural similarity to the human brain. Large animals also provide better models for testing with noninvasive imaging modalities (such as magnetic resonance imaging (MRI) and spectroscopy of disease processes, and positron emission tomography (PET) imaging of the activity of the reporter gene) as they are suitable for use with human clinical magnets and instruments (Wolfe et al., 2006). Another advantage of large animal models is their treatment and evaluation as individuals, facilitating assessments of the range of success and failure. At the same time, the production and evaluation of large statistically significant cohorts of affected animals and normal controls permit the study of variations in factors such as age at treatment and analysis, class of gene therapy vector, route of administration, and other variables. It would be virtually impossible to assemble the statistically significant cohorts or to treat and evaluate normal controls in humans. Also, human genetic disease groups are small but often harbor a variety of mutations, whereas genetically diseased animal breeding colonies typically have a unique mutation. Large animals in a colony thus provide a uniform genetic mutation that causes the disease, but at the same time, their genetic background is relatively outbred compared to inbred strains of mice. Another reason for translation from large animals to human clinical trials is that these live much longer than rodents, enabling studies on long-term effects of experimental gene therapy. The major limitations to the use of large animal models for biotherapeutic studies are high vivarium costs, long reproductive cycles, lack of some reagents, and few physiological differences from humans. Hence, some of the most successful studies have been complemented by mouse disease models (e.g., for hemophilias, lysosomal diseases). In these cases, the mouse model permits relatively fast progress to develop optimal vector designs and test proof of concept before testing in a large animal model of the best strategies for gene delivery to determine whether they produce similar outcomes, which can accelerate translation to clinical trials (Rawle and Lillicrap, 2004).

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11.9.2 Strategies, methods, and vectors for gene transfer There are two basic types of gene delivery systems: in vivo, involving direct vector injection into the body; and ex vivo, genetic modification of cells in culture followed by transplantation. Synthetic vectors, formulated completely from chemical and biochemical elements, are desirable because they can be made without cell systems. Despite a significant amount of ongoing research, they have still not been developed into practical gene delivery systems; thus, the use of viral vectors remains more likely for now. The most widely used viral vectors are based on retroviruses (RV), lentiviruses (LV), adenoviruses (Ad), adeno-associated viruses (AAV), and herpes simplex viruses (HSV). The RV and LV vectors have RNA genomes, require a reverse transcription step, and integrate into the host cell genome; the Ad, AAV, and HSV vectors have DNA genomes. The RV vectors require dividing cells to productively infect target cells, while the others can infect nonmitotic cells. It is possible to change the host range and cell tropism of most vectors by substituting alternate viral surface proteins, greatly improving the delivery specificity and range for viral vectors. Ex-vivo gene delivery involves the transplantation of genetically engineered cells in vitro, which expresses proteins used to treat pathological cells and also to deliver particular proteins. This form of gene therapy can be used to genetically engineer the somatic cells, which are derived from the affected individuals, altering them so that they express the deficient enzymes in order to avoid transplantation issues. This system has the distinctive properties, which allow for the regeneration of cells and allows for the migration of these cells throughout the system. Once stem cells (also known as progenitor cells) were discovered to be present in other tissues, ex-vivo therapy use was extended in other organs, such as the brain and liver. The drawbacks of ex-vivo therapy included the process of harvesting cells and then altering the genes in the culture before retransplanting them to the host was a difficult and tedious, and ex-vivo therapy resulted in the loss of the properties of stem cells. Once we discovered the process by which to genetically modify cells using direct injection, it eliminated the drawbacks of ex-vivo therapy, and expanding on this process has been extensively researched over the past decade. Now, induced pluripotent stem (iPS) cell methods have been discovered, using cells from easily accessible sources (e.g., skin fibroblasts or keratinocytes), and so the need for ex-vivo gene transfer has arisen. If we correct the genetic deficiency within the specific patient cells, the problems and side effects of cell transplantation no longer exist. However, there are limitations to using viral vectors. During the integration of the DNA into the host cell, the genetic changes have resulted in insertional mutagenesis and oncogenic transformation in clinical trials for X-linked SCID (Nienhuis et al., 2006). The host’s immune responses to the vector proteins, as well as the gene product (protein), have been a significant obstacle in achieving gene transfer for use in therapy, notably hemophilia (Murphy and High, 2008; Øvlisen et al., 2008). Occasionally, when the immune system recognizes the vector as a threat, upon repeat administration of a vector, there may be rejection, which results in the short-term correction of the flawed gene. Even though there are obstacles, large animal models show promising results and have shown that long-term expression of a missing gene is possible using therapy. The models of human genetic diseases in large animals have been important in the process of treatment in the approaches to clinical trials. The majority of genetic diseases do not have alternative therapy, and because of this, the importance of continuing to develop treatments for individual diseases is highlighted, even for rare and uncommon diseases. In large animal models of human cardiovascular genetic disease, gene therapy has been studied as a possible treatment, by Sleeper, Bish, and Sweeney from the University of Pennsylvania Veterinary School, USA. These animal models, which occur naturally, can be studied for human genetic heart diseases, offering the opportunity to understand and attempt potential therapies, including gene therapy. Although some of the diseases, e.g., patent ductus arteriosus, pulmonic stenosis would be difficult to treat with gene therapy, the ability to alter a large proportion of the myocardial cells will most likely make the various forms of inherited cardiomyopathy open to the transfer of a therapeutic gene. AAVs are likely the ideal vectors for cardiac gene therapy due to their low immunogenicity, allowing for stable expression of the transgene, a crucial factor when considering treatment of chronic cardiac disease. Large animal models available for the major forms of inherited cardiomyopathies (dilated cardiomyopathy, hypertrophic cardiomyopathy, and arrhythmogenic right ventricular cardiomyopathy) can help to study these conditions in humans. When studying juvenile dilated cardiomyopathy in Portuguese water dogs, scientists came up with an effective means to assess the process of therapeutic gene transfer, which allowed them to alter the course of cardiomyopathy and ultimately heart failure. By correcting the abnormal metabolic processes that cause heart failure (e.g., calcium metabolism, apoptosis), they are able to normalize diseased and faulty myocardial function. There are also various other diseases that have been treated in large animal models, such as diabetes, using gene therapy. Within a single treatment session, the dogs had recovered their health and no longer showed symptoms of diabetes. The treatment involves delivering two functional genes into the cells that allow the dog to sense and have a response to changes in blood sugar levels. The first gene is the insulin gene, which allows compensation for the low levels of insulin that causes the disease in most diabetics, and the second gene is the glucokinase gene, which allows for the detection of sugar levels. The two genes are delivered via viral vectors in nearly painless injections into the hind legs.

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After being delivered into the bloodstream, the vectors are absorbed into the animal muscle cells, where the genes proceed to integrate with the dog’s genome. As sugar levels increase, the glucokinase gene is activated, and this, in turn, activates the insulin gene, which begins the production of insulin. This brings down the blood sugar, just as it would in a normal healthy animal. This research needed to be done to find a more accurate model to treat diabetes, as the research had already previously been done in mice. For finding a more efficient model, dogs are often used for this type of research. After gene therapy, the dog’s blood sugar levels remained at healthy levels. The health of the dogs was then observed for 4 years without symptoms reoccurring and little to no side effects. These results are proof that gene therapy is successful and safe in large animals. The next phase of research will be clinical trials in people. Gene therapy using the same vector delivery system is licensed by the European Medicines Agency, and the wait will not be too long for clinical trials to begin. Gastroenterologists Selaru and Kumbhari at Johns Hopkins, have taken major steps in the direction of helping patients suffering from particular liver disorders utilizing an increasingly common endoscopic procedure, which delivers the therapeutic genes to the liver via the common bile duct. They believe that their method is safe and effective, and because of this, clinical trials in humans are very soon to be done. Selaru and Kumbhari describe a study which was performed on 12 pigs, regarding introducing therapeutic genes to the livers of the pigs via the bile ducts, accessing them by using an endoscopic technique most commonly used to diagnose, as well as treat problems in the gallbladder, biliary system, pancreas, and liver. The technique, known as endoscopic retrograde cholangiopancreatography (ERCP), was used to safely implant a human version of the functional genes into the cells of the pigs’ livers. The procedure was successful, and the engineered genes were expressing the intended proteins in all 12 of the animals. Tests were done on the 21st, 30th, and 60th days from the time of the procedures, and the animals all showed normal liver function. Usually, the administering of nonviral-based genes into the body is via intravascular injection, which has downsides, requiring large volume and cardiorespiratory risks. “In our study, we saw none of the side effects that accompany the intravascular injections,” Kumbhari mentioned. Since there was no biliary or liver injury, these results indicate that gene therapy via ERCP is much less invasive than injection. It is technically simpler and safer. The researchers used pigs as they provided the highest similarity to human patients, in terms of physiology, as well as genes. The ERCP method uses a flexible endoscope, accessing the common bile duct, which is located between the liver and the pancreas. The scope is inserted into the mouth of the anesthetized patient, and then the device is slowly guided into the passageway down the esophagus, into the stomach, and then to the duodenum. Now, a smaller device emerges from the end of the scope, which is guided by the endoscopist into the bile ducts. The procedure makes use of a camera on the endoscope and X-ray technology to observe the bile ducts. The trials, in this case, guided the injection of therapeutic genes into liver cells. Until now, it had not been possible to perform liver-specific hydrodynamic gene delivery in a large animal model with direct translatability to human trials. The technique was cumbersome, challenging, and invasive. There was very little progress in the direction of such clinical trials. One of the biggest challenges of venous injection of the genes that need to be administered has been the need for high volumes of the solution containing the engineered DNA molecules. This solution has to be pushed rapidly into veins, and this has led to ruptures in the walls, as well as other vein injuries, the DNA frequently misses the target site and fails to replicate successfully. The researchers at Johns Hopkins found that injection into the bile ducts requires a smaller volume of solution and does not injure the organs. Most importantly, the genes were successfully replicated and expressed their proteins. Of course, we can only hypothesize that this procedure will be equally benign in humans as it has been in our work with pigs, but it seems that safety will not be a barrier to clinical trials.

11.9.3 Gene therapy for the treatment of AIDS in animals AIDS is a dangerous disease of the immune system, which severely compromises the ability of the body to form any defense against invading pathogens. Researchers at the Lewis Katz School of Medicine LKSOM at Temple University and the University of Nebraska Medical Center (UNMC) have made great strides to treat the disease by eliminating replicationcompetent HIV-1 DNA from the genomes of living animals. This ability to counter the virus that causes AIDS highlights a major progression in the field of genetics. The study was reported in July 2019 in the journal “Nature Communications” and brought awareness of the ability to mediate the disease in animals, marking a crucial step toward developing a possible cure for human HIV infection. Kamel Khalili, Ph.D_., Laura H. Carnell, Professor and Chair of the Department of Neuroscience, Director of the Center for Neuro-virology, and Director of the Comprehensive NeuroAIDS Center at LKSOM said that their study showed that treatment to suppress HIV replication and gene editing therapy, when given sequentially, eliminated HIV from cells and organs of infected animals. HIV treatment used currently focuses on the use of antiretroviral therapy (ART). This method of therapy suppresses HIV replication but does not eliminate the virus from the body. This mediates the symptoms and reduces the increase of the virus. Therefore, ART is not a cure for HIV and requires life-long use. If it is stopped, HIV rebounds, renewing replication and fueling the development of AIDS. HIV rebounding

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is directly linked to the virus’s capability to integrate its genome into the DNA of the cells of the immune system, where it stays dormant and beyond the reach of antiretroviral drugs. In previous work, Khalili’s team used CRISPR-Cas9 technology to develop a gene editing and gene therapy delivery system, which was functioning by removing HIV DNA from the genomes, which harbored the virus. In rodents, it was shown that gene editing system could effectively excise and remove large fragments of HIV DNA from infected cells, which significantly impacted the expression of the viral genes. Unfortunately, this is not a viable method of complete treatment. LASER ART targets the integrated viral sequences and maintains HIV replication at low levels for extended periods of time, reducing the frequency required of ART. These long-affecting medications were made possible by pharmacological changes in the chemical structure of the antiretroviral drugs. The newer modified drugs were packaged into nanocrystals, which proceed to readily distribute to the select tissues where HIV is likely to be dormant. Then, the nanocrystals are stored within cells for weeks, slowly releasing the drug into the tissues. Based on this, the researchers utilized mice engineered to produce human T cells susceptible to HIV infection, which permitted the mice to undergo long-term viral infection and ART-induced latency. Then, as the infection was established, mice were treated with LASER ART and subsequently with the CRISPR-Cas9 gene replacement therapy. After the end of the treatment period, the mice were examined for viral presences. Analysis of the data revealed the complete elimination of HIV DNA in about one-third of the HIV-infected mice. The implications of this treatment are quite important, as once the treatment of AIDS can be translated from the animals to humans, the disease can be cured in humans. Epidermolysis bullosa is a condition where the skin is extremely sensitive and blisters easily, the mucous membranes breaking down simply through little contact. It is severe and can be mild to fatal because minor trauma and friction can cause blisters. At the University of Modena and Reggio, Dr. De Luca had been developing a way to counteract one of the mutations causing EB by insertion of a gene into the cells used for grafts. A 7-year-old child made an amazing recovery from this treatment, a method of gene therapy using a whole-body graft of genetically modified stem cells. This was the most ambitious attempt yet to treat a severe form of epidermolysis bullosa, which was most likely fatal, as the skin was weak and began to tear off at the slightest touch. This new approach, however, can address only a subset of the genetic mutations that cause EB. Nevertheless, the boy’s impressive recovery is very inspiring and has yielded positive results. This could yield insights that help researchers use stem cells to treat other genetic skin conditions, which may be debilitating or fatal. EB rises from mutations to any of various genes that code for proteins critical for anchoring the outer layer of skin, the epidermis, to the tissue below. This causes the missing or defective protein to stop holding the epidermis in place. Because of this, even slight friction or force can cause the skin to slough off from this minor damage, creating chronic injuries prone to infection. This condition would persist, even after the recovery of the injuries, because the healed epidermis would also be unable to bind to the lower levels of the skin, and therefore, would slough off again. According to Peter Marinkovich, a dermatologist at Stanford University, California, who specializes in treating EB patients, costs of bandages itself can be up to $100,000 a year. “They’re like walking burn victims,” he says. Their skin is so sensitive that the patients need to be wrapped in bandages thoroughly enough to not even put mild stress on the skin. Actually, the novel approach is similar to an established treatment for severe burns, in which layers of healthy skin are synthesized from a patient’s own cells and grafted over wounds. But stem cell biologist and physician Michele De Luca of the University of Modena and Reggio Emilia in Italy and his colleagues are working on developing a way to revert an EB-causing mutation by inserting a new gene into the cells utilized for grafts. Two EB patients have already been treated with this approach. They published encouraging results from their first attempt, where they grafted small patches of genecorrected skin on a patient’s legs in 2006. In 2015, De Luca’s team got an urgent request from doctors in Germany, whose 7 year old patient had junctional EB, a severe form of the disease, caused by a mutation in a gene encoding part of the protein laminin 332, which makes up a thin layer just below the epidermis. It was the very gene that De Luca’s team had been targeting in an ongoing clinical trial, but this case was dire. The boy was lacking most of his skin and had contracted multiple infections. This put him in a life-threatening septic state. This trial would be the first test of their gene therapy for such a large and intensely damaged skin area. De Luca’s team used a US postage-stamp-sized patch of skin from an unblistered part of the boy’s groin to culture epidermal cells, including stem cells that periodically regenerate the skin. They transformed the cells with a retrovirus bearing healthy copies of the target gene, LAMB3, and grew them into sheets ranging from 50 to 150 square centimetres. In two successive surgeries, a team at Ruhr University, Germany, covered the boy’s arms, legs, back, and some of his chest in the new skin. After a month, the researchers reported online in Nature that most of the new skin had started to regenerate, covering 80% of the patient’s body in the intact and elastic epidermis, and that he has not developed any blisters in the grafted areas in the 2 years since the treatment.

11.9.4 Brain cancer Cancer is a dangerous disease caused by faulty or mutated genes in the cells, depending on the part of the body whose cells are dividing at a rapid rate. A glioblastoma is a form of brain cancer where astrocyte cells in the brain undergo excessive division, and then they make their own blood supply to increase.

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An inducible, tumor-localized gene therapy has been tested for the first time in glioblastoma patients. The approach involves receiving an injection of an immune-activator gene into the brain tumor site and swallowing a pill that switches on the gene, resulted in the production of the activator interleukin 12 (IL-12), which causes infiltration of immune cells into tumor tissue. Glioblastoma is an aggressive and deadly form of brain cancer with an average survival post-diagnosis of just less than a year. Even with surgery to remove the tumor, followed by 6 weeks of chemoradiation, the tumor is back, as per neurosurgeon Antonio Chiocca of Harvard Medical School, who led the study. The bloodebrain barrier prevents a number of chemotherapeutics from reaching the tumor. Cancer itself is highly heterogeneous such that even if a drug is effective against certain cells, others may grow to take their place. Furthermore, glioblastomas can be described as “immunologically cold,” which means they create an environment that is immunosuppressive and limits the infiltration of cancerfighting immune cells. Converting a cold tumor into a hot treatable one is the strategy for novel therapeutic approaches. To do so, researchers have administered IL-12 proteinddescribed by Chiocca as a “powerful master regulator of the immune system.” But systemic IL-12 treatment can be toxic, causing flu-like symptoms, or in more serious cases, an inflammatory response known as cytokine release syndrome that can lead to organ failure. This harmful side effect is why the treatment still has to be monitored extensively. To maximize local levels of IL-12 while preventing high circulating levels, the team used an adenoviral vector carrying the IL-12 gene and injected it directly into tumor targets. The drug Veledimex could only activate the gene’s transcription, to control IL-12 dosage. If the patient stops taking the drug, the gene gets shut off again, and thus, translation of the gene coding for the IL-12 protein. This is similar to the induced operon regulated by glucose for the production of insulin. Veledimex crosses the blood-brain barrier, drives vector IL-12 expression, and shrinks tumors in murine glioblastoma. So Chiocca and colleagues tried the approach in 31 patients who were going through repeat surgery to remove regrown tumor tissue. During surgery, the patients were given injections of the vector into tissue surrounding the removed tumor. They then took varying daily doses (10, 20, 30, and 40 mg), increasing by 10 mg per day of Veledimex for up to 2 weeks. The blood levels of IL-12 and interferon-g, a cytokine produced in response to IL-12 were measured before, during, and after the course of Veledimex and were shown to rise with treatment and fall after cessation, with levels of the proteins correlating largely with dose. In tumor tissue removed from five patients who needed advanced surgery, more immune cells infiltrating the area were seen as compared with previously removed tumor tissue. The IL-12, interferon, and immune cell infiltration all suggest the approach is indeed switching on the gene and ramping up immunity (Chiocca et al., 2019).

11.9.5 The plastic bubble disease SCID is a rare condition, which affects the human immune system. It completely disables the immune system and leads to severe immunodeficiency. The patient has to endure living inside a plastic bubble to prevent exposure to pathogens. This disease mostly occurs in infants as the survival rate of individuals born with this disease is fairly low. Because of this, few adults have survived Immunodeficiency diseases. A pilot clinical study has shown that gene therapy restores the immune systems of infants with a lethal genetic disorder where infection-combating immune cells do not develop or function normally. Eight infants with the disorder, called X-linked severe combined immunodeficiency (X-SCID), received an experimental gene therapy codeveloped by NIH scientists. They experienced significant enhancements in immune system function and were growing normally up to 2 years after the treatment. The new strategy seems simpler, safer, and more effective than previously tested gene-therapy methods for X-SCID. Infants having X-SCID, as a result of mutations in the IL2RG gene, become highly susceptible to drastic infections. If left untreated, the disease is usually fatal. Infants with X-SCID are treated with transplants of blood-forming stem cells, ideally from a genetically compatible sibling. However, less than 20% of infants with the disease have such a donor as the criteria for compatible stem cells are very intensive. Those without a matched sibling generally receive transplants from a parent or other donor, which often only restore immunity in parts, since such patients require lifelong treatment and still keep experiencing complex medical problems, such as chronic infections and complete immune system shutdown. In patients with X-SCID, to restore immune function, scientists at NIAID and St. Jude Children’s Research Hospital in Memphis, inserted a normal copy of the IL2RG gene into the child’s own blood-forming stem cells as an experimental gene therapy strategy. The Phase 1/2 trial reported listed eight infants aged 2e14 months who were just diagnosed with X-SCID and lacked a sibling donor who may be genetically matched. The approach involves initially obtaining bloodforming stem cells from a patient’s bone marrow for gene therapy. Then, an engineered lentivirus that cannot cause illness is used as a vector to deliver the normal IL2RG gene to the cells. Finally, the stem cells are injected back into the patient, post a low dose of chemotherapy medication busulfan to help the genetically corrected stem cells get accepted and establish themselves in the bone marrow to begin producing new blood cells. In seven of the eight infants, normal numbers of various types of immune cells, such as T cells, B cells, and natural killer (NK) cells, developed within three to 4 months after gene therapy. While the eighth participant initially had low numbers of T cells, the numbers g increased following a second infusion of the LKSOM modified stem cells. Viral and

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bacterial infections that participants had before the treatment resolved afterward. The experimental gene therapy was safe overall, according to the researchers, although some participants experienced expected side effects such as a low platelet count following chemotherapy.

11.9.6 Cure for blindness With aging comes the degeneration of vision. By employing gene therapy, is there a way to cure this degeneration or even prevent it? Advances in diagnosis and analysis are now being used to find novel clinical solutions for blindness, as reviewed in the 10th Anniversary Series of Science Translational Medicine. Gene replacement or gene editing strategies could, in theory, reverse the loss of vision and lead to close to normal visual corrections. Intervention during the early stages of retinal degeneration, when the photoreceptor cells (rods and cones) are still intact, is particularly promising (Allocca et al., 2006; Stieger et al., 2009). The goal of Gene-independent strategies is to prevent or slow the progressive degeneration of photoreceptor cells with neuroprotective agents in a broad spectrum of retinal dystrophies. However, other options retinal prostheses, optogenetic therapy, and Stem cell therapy can restore vision after late retinal degeneration. These approaches can be applied independent of the underlying mutation and are expected to restore a small degree of vision in blind patients. Stem cell therapies to replace degenerated cells for restoring vision are under development or clinical evaluation in a wide range of degenerative retinal conditions. By expressing an optogenetic encoding a light-activated channel or pump in the remaining inner retinal cells, optogenetic therapy makes cells light-sensitive. It could be used to resensitize a degenerated retina to visible light independent of the photoreceptor cell loss, causing mutation. Specific traits make the eye fit for diagnostic and therapeutic intervention: easy access, small size, high compartmentalized internally, and cell populations that are stable. The optical transparency of the eye permits direct visualization with high-resolution imaging and precise evaluation of disease stage and response to therapy. The relative immune privilege of the eye, especially within the subretinal space, reduces adverse responses to injected vectors and gene products.

11.10 Some other potential uses of gene therapy The most interesting but complicated potential use of gene therapy is for human genetic engineering. If we could control the genetic material, it would be possible to alter the features of any human being, suffering from disease or not. Potential therapy can be done for treating syndromes that are not usually considered to be a disease. Higher or lower metabolism could be altered, down to the most basic of human functions. Gene doping is an experimental process by which athletes can improve their performance by altering their genes for better overall health and muscle development. This could have unknown side effects, as well as create an imbalance between players having undergone gene doping and those who have not. The implications of this ability to alter the physical appearance, mental faculties, metabolism, and essentially engineer qualities we desire into a human specimen are immense. Ethically, this is very ambiguous. When can parents be allowed to change the genes of their unborn child, to prevent a disease, or to prevent the likelihood of developing a disease? But it can also be abused to add beneficial or detrimental changes according to the parent’s desires. This has ethically negative implications, as it decides the child’s features and faculties for them. It could be used to selectively enhance offspring and lead to less genetic diversity.

11.11 Safety issues of gene therapy The scientific considerations for GTPs include the selection of appropriate gene delivery vector/modality for the disease/ tissue target, design of the expression cassette to ensure clinically relevant expression levels, specificity of gene expression to prevent unwanted side or off-target effects and to minimize host immune reactions. The design of preclinical and clinical studies for GTP differ significantly from the other chemical and biological drugs, because of the complexity of the vector interaction with the host cells wherein the effects of vector uptake into host cells, response of the host immune system, the outcome of the integration of genetic material into host chromosomes and levels of transgene expression from the host cells determine the final therapeutic efficiency. The GTP involves different components, such as the transgene cassette, the transgene regulatory systems, the delivery vectors, and the cellular component (in ex-vivo modifications). The scientific and ethical concerns for gene therapy primarily stem from the profound effect that genes exert on living cells by conferring novel properties and functions. The GTP ideally should not cause harm such as teratogenicity, excessive immune

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activation (e.g., aberrant CAR-T activity), introduction of unwanted mutations (off-target gene editing), or unwanted host immune response to GTP (neutralizing antibodies to AAV). Such gene augmentation techniques have the potential for misuse to get unnatural enhancement (in sports/defense sectors to increase physical performance) or to select for specific traits in newborns (designer babies by gene editing). All such applications are not allowed unless scientific or ethical justification can be provided, which is acceptable under socio-ethical norms and the laws of the country. Another danger is that the new gene might be inserted in the wrong location in the DNA, causing harmful mutations to the DNA or even cancer. This has occurred in clinical trials for X-SCID patients, using hematopoietic stem cells transduced with a corrective transgene using a retrovirus, and led to the development of T-cell leukemia in four of 20 patients. Also, when viruses are used to deliver DNA to cells into the patient’s body, there is a chance that this DNA could unintentionally affect the patient’s reproductive cells. In such a case, it could produce changes that may pass on if a patient has children after treatment. Additional concerns include the chance that the transferred genes could be induced too much, producing an excess of the missing protein and be harmful, and that the viral vector could cause an immune reaction, and the virus could be transmitted from the patient to another or into the environment. However, this basic mode of gene correction currently shows much promise, and doctors and scientists are working hard to foresee and fix any potential problems that could exist. They are using animal models for testing and other precautions to identify and avoid these risks before any clinical trials are conducted in humans. Despite a relatively new method to treat disease, studies have been done to observe the potential health risks, inflammation, and toxicity. Federal laws and regulations protect the patients involved in clinical trials, and thus, ensure oversight over the research being done. The FDA has the ability to reject or approve these clinical trials, and therefore, only the trials which are guaranteed as safe as approved. The NIH also gives guidelines for the universities and hospitals to follow for conducting clinical gene therapy trials to guarantee the safety of research in the field. Because of this, all trials must be registered with the NIH Office of Biotechnology Activities. The process of the trial is then reviewed by the NIH Recombinant DNA Advisory Committee (RAC) to determine whether it is safe, ethical, and plausible. Finally, an Institutional Review Board and Institutional Biosafety Committee have to approve the trials before it is performed. The Board consists of a committee of scientists, medical advisors, and consumers that review all the research conducted. The Biosafety Committee thoroughly reviews the safety hazards and potential unsafe studies. Due to these several levels of evaluation and review, the highest priority when conducting gene therapy trials is the safety of the patient.

11.11.1 Ethical and moral concerns surrounding gene therapy Even though Gene therapy is an expensive medical approach, moving it out of the laboratory and into clinical practice requires innovative financial schemes and regulatory policies, and along the way, clinicians, patients, and policymakers grapple with tricky ethical questions. The alteration of the basic blueprint and function of the body leads to many ethical concerns, which are unique to gene therapy and not any other form of medical research. It raises the concern of drawing the line between beneficial and gratuitous gene therapy. The traits which can be considered “normal” and the traits that are considered a disability or a disorder are difficult to distinguish. Current gene therapy treats the bone marrow and blood cells and is therefore localized to the individual and not passed onto the offspring. Germline gene therapy is forbidden as there is still no fixed rule for which conditions should be allowed to be treated and which should not. Conditions that are dangerous or harmful diseases can be treated in-vivo, but other things can also be altered the same way. The high price of the therapy excludes a vast majority of the populace from receiving the treatment. This is a temporary hurdle, and as scientific progress occurs and the techniques are improved, the prices of the treatment will reduce, and it will be widely available. This, however, comes with a plethora of other concerns. As the use of gene therapy becomes widespread, what happens to societal diversity and variation? Society could potentially become less accepting of those who are different. As it becomes more and more practiced, it could then be used to tinker with basic human traits such as looks, intelligence, or athletic ability leading to loss of genetically “perfect” humans as everyone is treated for their “disabilities.”

11.12 Conclusion In the future, gene therapy may enable doctors to treat a disorder by inserting a gene into a patient’s cells instead of employing drugs or surgery. However, there are several hurdles to overcome for Gene therapy to be effective: Immune responses by the body, multigenetic disorders, cost short-lived natures, and risk of side effects, including immune response to the working gene copy that has been inserted by causing inflammation. The corrected gene may get incorporated into the

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wrong position or might produce too much of the missing enzyme/protein, causing other health problems. There are also ethical complications arising from the nature of gene therapy itself, and as to what we decide to be normal traits versus undesirable ones. As gene therapy becomes more widespread, should we be allowed to choose the traits that we desire?

References Acland, G.M., Aguirre, G.D., Ray, J., Zhang, Q., Aleman, T.S., Cideciyan, A.V., Pearce-Kelling, S.E., Anand, V., Zeng, Y., Maguire, A.M., Jacobson, S.G., Hauswirth, W.W., Bennett, J., 2001. Gene therapy restores vision in a canine model of childhood blindness. Nat. Genet. 28 (1), 92e95. Allocca, M., Tessitore, A., Cotugno, G., Auricchio, A., 2006. AAV-mediated gene transfer for retinal diseases. Expet Opin. Biol. Ther. 6 (12), 1279e1294. Bauer Jr., T.R., Adler, R.L., Hickstein, D.D., 2009. Potential large animal models for gene therapy of human genetic diseases of immune and blood cell systems. ILAR J. 50 (2), 168e186. Berges, B.K., Wolfe, J.H., Fraser, N.W., 2007. Transduction of brain by herpes simplex virus vectors. Mol. Ther. 15 (1), 20e29. Casal, M., Haskins, M., 2006. Large animal models and gene therapy. Eur. J. Hum. Genet. 14 (3), 266e272. 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In: Rare Diseases and Orphan Products: Accelerating Research and Development. National Academies Press (US). Gagliardi, C., Bunnell, B.A., 2009. Large animal models of neurological disorders for gene therapy. ILAR J. 50 (2), 128e143. Ginn, S.L., Amaya, A.K., Alexander, I.E., Edelstein, M., Abedi, M.R., 2018. Gene therapy clinical trials worldwide to 2017: an update. J. Gene Med. 20 (5), 3015. Gollomp, K.L., Doshi, B.S., Arruda, V.R., 2019. Gene therapy for hemophilia: progress to date and challenges moving forward. Transfus. Apher. Sci. 58 (5), 602e612. Hasbrouck, N.C., High, K.A., 2008. AAV-mediated gene transfer for the treatment of hemophilia B: problems and prospects. Gene Ther. 15 (11), 870e875. Haskins, M., 2009. Gene therapy for lysosomal storage diseases (LSDs) in large animal models. ILAR J. 50 (2), 112e121. Horn, P.A., Morris, J.C., Neff, T., Kiem, H.P., 2004. Stem cell gene transfer–efficacy and safety in large animal studies. Mol. Ther. 10 (3), 417e431. Jiapaer, S., Furuta, T., Tanaka, S., Kitabayashi, T., Nakada, M., 2018. Potential strategies overcoming the temozolomide resistance for glioblastoma. Neurol. Med.-Chir. 58 (10), 405e421. Kang, S.M., Song, J.M., Kim, Y.C., 2012. Microneedle and mucosal delivery of influenza vaccines. Expert Rev. Vaccines 11 (5), 547e560. Koeberl, D.D., Pinto, C., Brown, T., Chen, Y.T., 2009. Gene therapy for inherited metabolic disorders in companion animals. ILAR J. 50 (2), 122e127. Leber, K.J., 2008. Congenital amaurosis: from darkness to spotlight. Ophthalmic Genet. 29 (3), 92e98. Lee, E.S., Jin, S.Y., Kang, B.K., Jung, Y.T., 2019. Construction of replication-competent oncolytic retroviral vectors expressing R peptide-truncated 10A1 envelope glycoprotein. J. Virol. Methods 268, 32e36. https://doi.org/10.1016/j.jviromet.2019.03.008. Logg, C.R., Logg, A., Tai, C.K., Cannon, P.M., Kasahara, N., 2001. Genomic stability of murine leukemia viruses containing insertions at the Env-30 untranslated region boundary. J. Virol. 75, 6989e6998. Malech, H.L., Hickstein, D.D., 2007. Genetics, biology and clinical management of myeloid cell primary immune deficiencies: chronic granulomatous disease and leukocyte adhesion deficiency. Curr. Opin. Hematol. 14 (1), 29e36. Mavilio, F., Ferrari, G., 2008. Genetic modification of somatic stem cells. The progress, problems and prospects of a new therapeutic technology. EMBO Rep. S1, S64eS69. McConnell, M.J., Imperiale, M.J., 2004. Biology of adenovirus and its use as a vector for gene therapy. Hum. Gene Ther. 15 (11), 1022e1033. Murphy, S.L., High, K.A., 2008. Gene therapy for haemophilia. Br. J. Haematol. 140 (5), 479e487. Nichols, T.C., Dillow, A.M., Franck, H.W., Merricks, E.P., Raymer, R.A., Bellinger, D.A., Arruda, V.R., High, K.A., 2009. Protein replacement therapy and gene transfer in canine models of hemophilia A, hemophilia B, von willebrand disease, and factor VII deficiency. ILAR J. 50 (2), 144e167. Nienhuis, A.W., Dunbar, C.E., Sorrentino, B.P., 2006. Genotoxicity of retroviral integration in hematopoietic cells. Mol. Ther. 13 (6), 1031e1049. Oehmig, A., Fraefel, C., Breakefield, X.O., 2004. Update on herpesvirus amplicon vectors. Mol. Ther. 10 (4), 630e643. Øvlisen, K., Kristensen, A.T., Tranholm, M., 2008. In vivo models of haemophilia e status on current knowledge of clinical phenotypes and therapeutic interventions. Haemophilia 14 (2), 248e259. Patterson, D.F., Haskins, M.E., Jezyk, P.F., Giger, U., Meyers-Wallen, V.N., Aguirre, G., Fyfe, J.C., Wolfe, J.H., 1988. Research on genetic diseases: reciprocal benefits to animals and man. JAVMA 193, 1131e1144. Philpott, N.J., Thrasher, A.J., 2007. Use of non-integrating lentiviral vectors for gene therapy. Hum. Gene Ther. 18 (6), 483e489.

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Pierson, T.M., Wolfe, J.H., 2005. Gene therapy for inherited diseases of the central nervous system. In: Lynch, D. (Ed.), Neurogenetics: Scientific and Clinical Advances. Marcel Dekker, New York, pp. 43e85. Rawle, F.E., Lillicrap, D., 2004. Preclinical animal models for hemophilia gene therapy: predictive value and limitations. Semin. Thromb. Hemost. 30 (2), 205e213. Rogers, C.S., Stoltz, D.A., Meyerholz, D.K., Ostedgaard, L.S., Rokhlina, T., Taft, P.J., Rogan, M.P., Pezzulo, A.A., Karp, P., Itani, O., Kabel, A., Wohlford-Lenane, C., Davis, G., Hanfl, R., Smith, T., Samuel, M., Wax, D., Murphy, C., Rieke, A., Whitworth, K., Starner, T., Brogden, K., Shilyansky, J., McCray, P.B., Zabner, J., Prather, R., Welsh, M., 2008. Disruption of the CFTR gene produces a model of cystic fibrosis in newborn pigs. Science 321, 1837e1841. Rosenberg, S.A., Aebersold, P., Cornetta, K., Kasid, A., Morgan, R.A., Moen, R., Karson, E.M., Lotze, M.T., Yang, J.C., Topalian, S.L., 1990. Gene transfer into humans-immunotherapy of patients with advanced melanoma, using tumor-infiltrating lymphocytes modified by retroviral gene transduction. N. Engl. J. Med. 323 (9), 570e578. Sanjana, N.E., Cong, L., Zhou, Y., Cunniff, M.M., Feng, G., Zhang, F., 2012. A transcription activator-like effector toolbox for genome engineering. Nat. Protoc 5 (1), 171e192. Schultz, B.R., Chamberlain, J.S., 2008. Recombinant adeno-associated virus transduction and integration. Mol. Ther. 16, 1189e1199. Segal, B.H., Veys, P., Malech, H., Cowan, M.H., 2011. Chronic granulomatous disease: lessons from a rare disorder. Biol. Blood Marrow Transplant. 17 (1), S123eS131. Sleeper, M.M., Bish, L.T., Sweeney, H.L., 2009. Gene therapy in large animal models of human cardiovascular genetic disease. ILAR J. 50, 199e205. Stieger, K., Lhériteau, E., Moullier, P., Rolling, F., 2009. AAV-mediated gene therapy for retinal disorders in large animal models. ILAR J. 50, 206e224. Tabin, C.J., Hoffmann, J.W., Goff, S.P., Weinberg, R.A., 1982. Adaptation of a retrovirus as a eucaryotic vector transmitting the herpes simplex virus thymidine kinase gene. Mol. Cell Biol. 4, 426e436. Vite, C.H., Niogi, S.N., McGowan, J.C., Passini, M.A., Drobatz, K.J., Haskins, M.E., Wolfe, J.H., 2005. Effective gene therapy for an inherited diffuse CNS disease in a large animal model. Ann. Neurol. 57, 355e364. Wang, Z., Chamberlain, J.S., Tapscott, S.J., Storb, R., 2009. Gene therapy in large animal models of muscular dystrophy. ILAR J. 50, 187e198. Wang, Y., Zhou, X.Y., Xiang, P.Y., Wang, L.L., Tang, H., Xie, F., Li, L., Wei, H., 2014. The meganuclease I-SceI containing nuclear localization signal (NLS-I-SceI) efficiently mediated mammalian germline transgenesis via embryo cytoplasmic microinjection. PloS One 9 (9), 108347. Wolfe, J.H., Acton, P., Poptani, H., Vite, C.H., M.G., M.G., 2006. Molecular imaging of gene therapy for neurogenetic diseases. In: Kaplitt (Ed.), Gene Therapy in the Central Nervous System: From Bench to Bedside. Academic Press, San Diego, pp. 335e350. Wu, Z., Asokan, A., Samulski, R.J., 2006. Adeno-associated virus serotypes: vector toolkit for human gene therapy. Mol. Ther. 14, 316e327. Yang, S.H., Cheng, P.H., Banta, H., Piotrowska-Nitsche, K., Yang, J.J., Cheng, E.C., Snyder, B., Larkin, K., Liu, J., Orkin, J., Fang, Z.H., Smith, Y., Bachevalier, J., Zola, S.M., Li, S.H., Li, X.J., Chan, A.W., 2008. Toward a transgenic model of Huntington’s disease in a non-human primate. Nature 453, 921e924.

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Chapter 12

Nanobiotechnology in animal production and health Ravindra Pratap Singh and Kshitij R.B. Singh Department of Biotechnology, Indira Gandhi National Tribal University, Amarkantak, Madhya Pradesh, India

12.1 Introduction Nanotechnology (NT) is a science that deals at the nanoscale that is between 1 and 100 nm. It is able to see and control atoms and molecules by scanning tunneling microscope (STM) and Atomic force microscopy (AFM). Nanomaterial integrated biology is known as nanobiotechnology (NBT) and has the capability for diagnostics and therapeutics using a variety of nanomaterials; for examples, metal and metal oxides nanoparticles (NPs), quantum dots (QDs), carbon-based NPs, magnetic NPs, liposomal NPs, polymeric NPs, and dendrimer NPs. NBT is extending its applications for animal production and health, as it has revolutionized agriculture and livestock production by advancing animal rearing and their feed management. In addition, NBT offers scientific advantages pertaining to nanomaterials bioavailability and biodegradability, which help counter the economic losses, not only to livestock animals but also to their health-related improvement; further, it requires more R&D toward technical, societal, and policy for adopting regulations to control timely implications of this emerging domain. The animal welfare, the safety of feed products, i.e., livestock production and environmental health risks, are the main cause of concerns for the researchers of biotechnology and NBT. Prior studies reported the use of nanofluidic systems for the mass production of animal embryos, drug delivery of smart drug and biosensors/nanobiosensors to maintain the health of livestock by detection of toxic compounds and pathogens within no time (Arora et al., 2006; Singh et al., 2008, 2009a, 2010, 2011a; Singh and Pandey, 2011). Genomics is the study of the whole genetic material of an organism known as the genome, the prospects of genomics are the necessity of current scenario which is to be highlighted toward human but also for animal disease prevention, treatment, drug development, and as well as in wildlife conservation. The mapping of animal genome is significant work, which identifies the desired gene sequences of sheep, pig, poultry, and livestock, which can improve the quality and health of animals. The disease resistance, production of meat, and dairy product could be improved using gene probes on biochips, breeders/bioreactors, which can further screen out animal diseases based on genes. Keener et al. (2004) reported the recent development of nanotechnological tools and techniques used for animal production and health to solve the problems in poultry infections caused by Campylobacter. Guo et al. (2007) reported Cu (II)-exchanged montmorillonite nanoparticles (MMT-Cu) as an antimicrobial agent in the diet to increase growth performance (weight gain), digestive function (digestibility) of weaned pigs. Nano-sized minerals and vitamin diet as animal feed can solve the problem of early diagnosis of animal diseases. The smart treatment strategy was reported using drugs and nutraceuticals to improve animal feeds. Mycotoxicosis has been reported due to the consumption of animal feeds, which are poorly stored; as a result, huge economic losses occurred in the agriculture sector, reduced nutritional value in animal feed, as well as decreased animal meat production, and dairy products (Bryden, 2012). Zavareh et al. (2015) reported inorganic arsenic (As-III) in drinking water sources which possess human health hazard. Makwana et al. (2014) reported the use of cinnamaldehyde (derived from cinnamon) as a food antimicrobial, as well as a flavoring agent, which, in turn, can have immense applications in food safety. Further, they have also demonstrated encapsulated liposomes cinnamaldehyde in an active food packaging of liquid foods. Nanomaterials are used as antiviral agents and also for the detection of viruses using nanobiosensor, which can improve animal production and health. They can also detect environmental and clinical analytes of interest, for example, hormone levels, and metabolites (Singh et al., 2009b; Singh, 2011a,b, 2016). Furthermore, nanomaterials are used in

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cryopreservation of testis, ovary, sperm, oocytes, embryos (Sagadevan and Periasamy, 2014), and toward animal production for good quality of meat products that are free from pathogens and contaminants (Lee et al., 2011).

12.2 Quantum dot nanoparticles The products and processes based on Quantum dot nanoparticles are beneficial not only for animal production and health but also for other significant applications, as shown in Fig. 12.1. QDs are able to improve sperm motility and ova movement (Feugang et al., 2012). Furthermore, the biocompatible and bioluminescent QDs NPs are able to track the gametes of the male pig (Feugang et al., 2015). Weldon et al. (2018) reported nanomaterials in consumer/commercial products, which are posing potential toxicity, for example, cadmium-selenium containing quantum dots (QDs) and suggested an immediate need for health risk assessments in vitro and in vivo. Tao et al. (2015) reported cytosine-phosphate-guanine QDs as a promising optical probe, which showed excellent cellular uptake efficiency. Li et al. (2018) reported the diagnosis and treatment of communicable diseases using aptamer-modified fluorescent-magnetic multifunctional nanoprobes. Stanisavljevic et al. (2014) reported CdTe QDs (2 nm size) interaction with chicken genomic DNA. The genome sequence data of humans and pathogens are utilized as biomarkers for early diagnosis of cancer disease. The proteomics integrated with nanotechnology is known as nanoproteomics, which use diverse types of nanomaterials like QDs, Gold (Au) NPs, and Carbon nanotubes (CNTs). The production of nanomaterials, either commercially or industrially, has stimulated concerns relating to their environmental risks on animal production and health (Singh et al., 2012b). Ray et al. (2011) reported reproductive toxicity due to engineered nanoparticle usage in animal germ cells and embryos. Ludwig et al. (2014) reported anti-rbST antibodies (rbST biomarker) labeled quantum dot (QD) based cellphone to detect antirecombinant bovine somatotropin antibodies in animal milk. Rodriguez-Fragoso et al. (2012) reported maltodextrin coated cadmium sulfide semiconductor nanoparticles (QDs) is showed cytotoxic and embryotoxic to chicken embryos.

12.3 Carbon-based nanoparticles Carbon Nanoparticles (CNPs) have shown low toxicity and are used for diagnostics and as drug carriers for therapeutics; different types of CNPs are graphite, nanodiamond, fullerenes, graphene, and CNTs but biodegradability is the main cause of concern for their utility (Lamprecht et al., 2009). Fig. 12.2 shows the broad-spectrum applications of carbon-based nanoparticles.

FIGURE 12.1 Schematic presentation of quantum dot nanoparticles applications.

FIGURE 12.2 Shows wide applications of carbon-based nanoparticles.

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Mycotoxins are the main culprits of domestic animal diseases. Gibson et al. (2011) reported several mycotoxins such as aflatoxin-B1 and ochratoxin-A immobilized on nanodiamond substrates for detection and removal, respectively. Graphite is a carbon allotrope. Parvongnukul and Lumb (1978) reported hip prostheses coated by polytetrafluoroethylene-graphite for the use in animals. Further, Graphite-furnace based atomic absorption spectroscopy was used to determine the heavy metals like lead, cadmium, and selenium in blood of cow, sheep, goat, and in the milk of buffalo (Rahimi, 2013). Graphene is a carbon allotrope which is widely used in drug delivery and cellular imaging. Lee et al. (2015) reported treatment and prevention of diseases using reduced graphene oxide (rGO) coated polydimethylsiloxane film, whereas Ye et al. (2015) reported treatment and prevention of pseudorabies virus and porcine diarrhea virus using graphene oxide. Fullerene (C60) is a carbon allotrope, which is used in animals for disease prevention (Taylor et al., 1990). Fullerene-based compound, for example, shungite is been used as an antioxidant and antiinflammatory agent in health care (Sajo et al., 2017). CNT is also a carbon allotrope, which is used as an antibacterial agent in animal breeding. Carbon nanotube implantation to the skin is used to monitor the level of beta-estradiol in the blood to track the oestrus cycle in animals by near-infrared fluorescence method, and this, in turn, improves the production of animals. Microfluidics method is used for in vitro fertilization in animal breeding for better animal production, which maintains their health. In addition to theadvancement of human genome mapping, investigators are trying to identify gene sequences to improve the production and health of animals (O’Connell et al., 2002). Biochips are important silicon circuit-based smart devices to detect pathogens in diseases (e.g., check avian flu and mad cow disease) and animal feeds. Ahmad et al. (2019) reported the removal of these organic micropollutants (OMPs) consisting of polyaromatic hydrocarbon, antibiotics, and pesticides in water bodies by utilization of CNTs. Further, Facciolà et al. (2019) reported CNTs which shows cytotoxicity of neurons and impairing molecular pathways responsible for lower intelligence quotient (IQ) in children, and neurodegenerative diseases. In addition, CNTs are used as nanocarriers for the drug delivery and in contrast agents for imaging. Duke and Bonner (2018) reported that CNTs are responsible for pulmonary fibrosis, production of oxidative stress, and death in animals. Castranova et al. (2013) reported CNT’s application for targeted drug delivery, bone grafting, and dental implants. Govindasamy et al. (2017) reported estimation of chloramphenicol (in milk and honey) based on molybdenum disulfide nanosheets coated with multi-walled carbon nanotubes and single-wall carbon nanotubes which are used as a drug carrier containing an antiviral drug to treat viral diseases. Mastitis in cows caused by bacteria was treated with various carbon nanoparticles, graphene oxide (GO), rGO, silver (Ag) NPs, and GO-Ag NPs nanocomposites (Zhang et al., 2016). Manjunatha et al. (2018) reported the toxicity of pristine graphene (pG) in the animal model (zebrafish embryo) and found cardiotoxicity and apoptosis, which suggest that this pG should be checked and controlled in a way that they do not get released in the environment. The health threat due to endocrine-disrupting chemicals (e.g., bisphenol-A (BPA)) could hamper the hormone biosynthesis, metabolism, homeostatic control, and reproduction. Detection and removal of toxic BPA are possible on either GO or CNTs-based nanobiosensor. BPA can mimic estrogen and could lead to bad health effects on animals, wildlife, and human beings. BPA is present in wastewater, groundwater, surface water, and drinking water (Singh et al., 2011a).

12.4 Dendrimers nanoparticles Infectious diseases are appearing due to microorganisms and transmitted by insects, plants, soil, and water; as a result, they reduce the immunity of the animals. Improved services of healthcare, nutrition, and better education may decrease the death of animals due to infectious diseases. Infectious diseases are treated by therapeutics like antibiotics, antiparasitic, antiviral, and antifungal agents but somewhat suffer from limitations, for instance, drug resistance problem, toxicity, and mode of administration. Therefore, targeted drug delivery utilizing nanocarrier to treat infectious diseases is the present scenario. These nanocarriers are QDs, dendrimers, carbon-based nanoparticles, liposomes, and polymer-drug conjugates. They have reduced the drug toxicity, enhanced the bioavailability/biocompatibility/biodegradability, solubility, and are nonimmunogenic; these unique properties can help to overcome drug resistance (Mintzer et al., 2012). Dendrimers are man-made polymers (Chahal et al., 2016), and have a variety of biomedical applications, as shown in Fig. 12.3.

FIGURE 12.3 Represents potential applications of dendrimers.

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Poly-L-lysine dendrimer has two primary amines due to the reason that it is biocompatible, flexible, biodegradable, and water-soluble; these dendrimers can acts as a gene carrier for the therapeutic purpose whereas Poly (propylene imine) dendrimer has 1; 4-diaminobutane or ethylenediamine core and branching unit consist of propylene imine monomer, which offers it diagnostic applications (Yandrapu et al., 2013; Singh et al., 2010). Thus, dendrimers are the best nanocarrier used in drug delivery systems to treat viral and parasitic infections by reducing the toxicity and targeting to kill the parasite. They are used as vaccine carriers for the delivery of vaccines to prevent schistosomiasis infection and increase the production of interleukin-2 and interferon. They also block the HIV (human immunodeficiency virus)-1 and check the hepatitis C infection. However, there is no data related to these on modes of mechanism. Thus, it is suggested that the study in this direction to determine mode of mechanism data is required. Leishmaniasis is a tropical disease caused by Leishmania parasite transmitted by female sand flies across the world (Ejazi and Ali, 2013). Leishmaniasis was treated earlier using sodium stibogluconate, meglumine antimoniate, but they developed resistance along with cardiotoxicity and pancreatitis. For overcominge this problem, amphotericin B, Miltefosine, and paromomycin are used as an alternative drug. Amphotericin B is an antifungal and antiparasite agent, and Miltefosine is an anticancer drug, but they are approved as oral drugs for leishmaniasis with limitations like low blood platelets, nephrotoxicity, diarrhea, etc. Furthermore, dendrimer, as a nanocarrier, is used as a super alternative to reduce not only toxicity of the drug but also to enhance drug solubility, and its degradability for the better treatment of leishmaniasis (Menezes et al., 2015). Jain et al. (2015a) reported a poly (propylene imine) dendrimers/mannose loaded with amphotericin B to treat leishmaniasis. Furthermore, Jain et al. (2015b) reported muramyl dipeptide conjugated with poly (propyleneimine) dendrimers loaded with amphotericin B, which was active against the parasite infection using macrophage cell lines and balb/c mice and formulations showed antileishmanial activity. Schistosomiasis is the parasitic disease caused by Schistosoma and transmitted by a fluke across the globe (Gryseels et al., 2006). Schistosomiasis was treated earlier with praziquantel but not effective well. For overcoming this problem, dendrimers have been used as a nanocarrier for drug intervention to eliminate the disease. Wang et al. (2014) reported dendrimer PAMAM (Poly(amidoamine))-Lys/SjC23 DNA vaccine for the prevention of Schistosoma japonicum infection, which enhances the immunoreactivity of this DNA vaccine. Toxoplasmosis is due to a parasite Toxoplasma gondii from the exposure to infected cat feces, eating undercooked meat, and also from contaminated water (Cook et al., 2015). Toxoplasma gondii parasite causes morbidity and mortality, and this can be treated by pyrimethamine and sulfadoxine but has few limitations in their use like toxicity, hypersensitivity, and it does not eliminate the parasite. However, peptide dendrimers are reported in the treatment of toxoplasmosis. Prieto et al. (2006) reported sulfadoxine-cationic dendrimer sulfadiazine as an effective drug for the treatment of toxoplasmosis. Lai et al. (2012) reported phosphorodiamidate morpholino oligomers with transductive peptide conjugate for the reduction of Toxoplasma gondii infections. Malaria is life-threatening, and half of the world’s population is at high risk of malaria transmission. Chloroquine, primaquine, and artemisinin are the desired drugs for the treatment of malaria, but they are somewhat resistant and produce toxicity (Hartman et al., 2010). For overcoming this problem, dendrimer nanocarriers are used because loaded desired drugs show better solubility, biocompatibility, and biodegradability (Taylor et al., 2012). Movellan et al. (2014) reported amphiphilic dimethylolpropionic acid (Bis-MPA) derivatives-based dendrimers with loaded antimalarial drugs like chloroquine and primaquine for the better and effective treatment of malaria. Agrawal et al. (2007) reported glycoconjugated peptide dendrimer-based chloroquine phosphate, coated and uncoated with poly-L-lysine dendrimers for the treatment of malaria; the result showed no toxicity and less immunogenic. HIV, meningitis, hepatitis, influenza, cervical cancer (human papillomavirus (HPV)), and herpes are viral diseases (Pritchard et al., 2015). Acquired immunodeficiency syndrome (AIDS) due to HIV infections has caused high death rates across the globe, and there is a serious need to develop a new drug to treat AIDS. Antiretroviral drugs are useful only for delaying the HIV infection and reducing the mortality rate but could not cure the HIV patients, and thus there is a need for a new treatment strategy. In this context, dendrimers can be used as a nanocarrier to be functionalized/encapsulated with an antiretroviral drug, which can improve the drug efficacy, and less drug toxicity (De Clercq, 2012). Zidovudine, an antiretroviral drug against HIV virus infection, was reported with few limitations like poor bioavailability, toxicity, and resistance. For overcoming these problems zidovudine was encapsulated using dendrimers for better results than alone. Vacas-Cordoba et al. (2014) reported a polyanionic carbosilane dendrimer (e.g., G3-S16 and G2-NF16) encapsulated zidovudine, efavirenz, and tenofovir and tested against X4 and R5 HIV-1 strains in vitro, the results of these studies showed a synergism. Type-A flu viruses are very detrimental to humans, animals, and wild birds, whereas type B virus has a less severe effect on humans, and influenza type C is less common (Robison et al., 2017). Influenza is treated with antiviral drugs, for example, amantadine oseltamivir, and rimantadine; influenza is resistant to these drugs. To overcome this problem of drug resistance, Landers et al. (2002) reported sialic acid-conjugated dendrimer polymers, and their result signifies less toxicity, and these conjugates were very effective to prevent influenza pneumonitis.

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Herpes simplex (Herpes simplex Virus (HSV)-1 and HSV-2) is sexually transmitted infections (STIs) and infected via saliva and skin across the globe (Chen et al., 2017). Traditional drugs are not effective and have developed resistance with toxicity so that to improve drug efficacy, dendrimer nanocarrier is used to kill the virus (Bastien et al., 2012). Luganini et al. (2011) reported acyclovir-peptide-dendrimers and its derivatives to kill HSV-1 and HSV-2 in Vero cells infected with HSV. Carberry et al. (2012) reported membrane-peptide gH (gH625) functionalized dendrimer derived from HSV-1 to prevent herpes virus infection effectively without any cellular toxicity. Cena-Diez et al. (2016) reported peptide carbosilane dendrimers, PAMAM dendrimers, and polysulfated galactose functionalized glycodendrimers to prevent sexually transmitted infections. Hepatitis A, B, and C are viruses, which cause liver infection (Ebert et al., 2015). It is chronic liver disease, and no vaccine against hepatitis C is available (Shivkumar et al., 2012). The sofosbuvir and ribavirin are antiviral drugs, which are used against hepatitis C infection. Dendrimers loaded antiviral drugs are useful (Heegaard et al., 2010). Sepulveda-Crespo et al. (2017) reported polyanionic carbosilane dendrimers to treat hepatitis C virus infection. Khosravy et al. (2014) reported dendrimer conjugated hepatitis B virus surface antigen (HBsAg) in vivo to treat the hepatitis infections. Lakshminarayanan et al. (2015) reported liver-targeted dendritic nano-vector functionalized with a galactopyranoside ligand for the delivery of siRNA for the treatment of hepatitis infections. Cervical cancer might be caused due to HPV (Remschmidt et al., 2013). It can be treated with chemotherapy, radiotherapy, and surgery in its early stage. Drugs like cisplatin, paclitaxel, and topotecan have shown side effects in terms of its resistance, and toxicity (Lorusso et al., 2014). Dendrimers can reduce immunogenicity, as well as the toxicity of peptide-based vaccines against cervical cancer (Kesharwani et al., 2015). Liu et al. (2013) reported peptide dendrimers to prevent HPV. Other examples were reported, such as multiantigenic peptide-polymer conjugates, PAMAM dendrimers conjugated with doxorubicin and dendrosome-based siRNA (Mekuria et al., 2016).

12.5 Liposomes nanoparticles Liposomes are colloidal structures and spherical vesicles made up of phospholipid’s bilayer. It shows permeability and charge density, along with encapsulation capability to load both hydrophilic and lipophilic drugs in an aqueous phase. In addition to these properties, it also exhibits biocompatibility, biodegradability, and low toxicity. Liposome nanoparticles are important for the wide range of applications, as shown in Fig. 12.4. Malam et al. (2009) reported Liposome mediated drug delivery for the multidrug-resistant cancers to prevent and treat the diseases. Sallovitz et al. (1998) reported liposomal formulations for the prevention of bacterial animal diseases. MacLeod and Prescott (1988) reported gentamicin entrapped liposome to treat mastitis in lactating cows. Leenders and de Marie (1996) reported lipid formulations entrapped amphotericin-B to treat fungal infections. Makabi-Panzu et al. (1994) reported liposomal formulation entrapped ribavirin and 20 30 -dideoxycytidine to treat viral diseases in animals. Rhee et al. (2012) reported hemagglutinin-derived synthetic peptide DNA-liposome nanoformulation to kill the influenza virus. Miao et al. (2017) reported a liposomal rabies vaccine in BALB/c mice and showed an immunopotentiator. Tada et al. (2018) reported nasal vaccination using pneumococcal surface protein A, nanoformulated with 1, 2-dioleoyl-3-trimethylammonium-propane

FIGURE 12.4 Shows biomedical applications of liposome nanoparticles.

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(DOTAP) and cholesteryl 3-N-(dimethylaminoethyl)-carbamate (DC-chol) liposomal complex. DOTAP/DC-chol liposome vaccine prevents pneumococcal infection. Nagao et al. (2016a) reported CO-bound HbV as a drug to treat colitis but also a variety of other disorders.

12.6 Metal and metal oxides nanoparticles Metallic NPs are in a variety of forms such as nanoparticles (zero-dimension), nanowires, and rods (1-dimension), nanosheet (2-dimension), and nanostructures (3-dimension). They are frequently used in a number of applications, as shown in Fig. 12.5. The metallic NPs can be synthesized (physicochemical and biological approaches) and can be modified with desired functional groups to bind with bioactive constituents (Shukla et al., 2010; Singh et al., 2012a; Singh, 2017, 2019a). Enormous properties of these NPs make it an interesting agent to overcome drug resistance, which is a life-threatening issue, and it also exhibits a variety of applications toward animal production and health. AgNPs are used in diagnosis, treatment, wound dressings, medical devices, and contraceptive devices, as an antibacterial, antiviral, antifungal, antiinflammatory, anticancer, and antiangiogenesis element. The NPs used in animal production are for the development of sensors/nanobiosensors, imaging, drug delivery, and tissue engineering, which is very important. Biologically synthesized AgNPs loaded vaccine injected into the mice and dogs. Thus, AgNPs-loaded rabies vaccines were established as a veterinary vaccine (Stankic et al., 2016). Antibiotic-resistant bacteria are detrimental to animal production and health. For overcoming this problem, combined therapy is an appropriate approach, which utilizes antibiotics and AgNPs to enhance the antibacterial activity with no toxicity (Gurunathan et al., 2014, 2015). Smekalova et al. (2016) reported gentamicin with AgNPs to kill the Actinobacillus pleuropneumoniae. Yuan et al. (2017) reported AgNPs to treat mastitis in goats. Fondevila et al. (2009) reported AgNPs to reduce remarkably coliform bacteria in weaned piglets. Gholami-Ahangaran and Zia-Jahromi (2013) reported nanosilver in the experimental aflatoxicosis in broiler chickens and showed complete removal of aflatoxin. Pineda et al. (2012) reported AgNPs as an antimicrobial growth-promoter supplement for broiler chickens via drinking water using immunoglobulin (IgG). Bhanja et al. (2015) reported AgNPs administration to chicken embryos and suggested that either alone AgNPs or in combination with amino acids enhance the immunity in chicken. However, Asgary et al. (2016) reported AgNPs as adjuvants in rabies vaccine and showed no toxicity. Gurunathan et al. (2018) reported AgNPs as an antibacterial agent to treat endometritis in cattle. AuNPs have a wide range of biomedical applications because of their good biocompatibility, easy surface functionalizations; they are being used as agents for imaging, cell tracking, biological sensing, drug delivery, and photothermal therapy. Nurulfiza et al. (2011) reported virus antibodies coated with AuNPs on strip to detect virus of bursal disease in

FIGURE 12.5 Shows important applications of metal and metal oxide nanoparticles.

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chickens. Wang et al. (2013) reported AuNPs to diagnose viral infections in pigs. Jiang et al. (2015) reported AuNPs to treat Toxoplasma gondii infection in dogs and cats. Fent et al. (2009) reported gum arabic, and maltose stabilized AuNPs to determine the tissue distribution profile. Chanda et al. (2014) reported AuNPs utilization as contrasting agent for tumor imaging in mice and dog models. Scharf et al. (2015) reported superparamagnetic iron oxide nanoparticle for cell-tracking in an ovine model caused tendonitis in sheep. Edge et al. (2016) reported dimercaptosuccinic acid-coated superparamagnetic iron oxide nanoparticles for the diagnosis and treatment of cancer in pig models. Zinc oxide (ZnO) NPs were reported and used as wound healing, antineoplastic, antibacterial, food preservative, as a feed additive, and angiogenic properties (Raguvaran et al., 2015). Mastitis was reported in high yielding animals due to infection by Staphylococcus, Streptococcus, and E. coli, and reduces the milk (Erskine et al., 2003). Several investigators reported that ZnO NPs treat the neoplastic disorder in domestic animals like mules, horses, and donkeys (Dobson et al., 2002). Salama et al. (2003) reported a zinc-methionine diet for milk production in dairy goats. Mishra et al. (2014) reported ZnO-NP for enhanced growth, development, and increase feed utility economically in weaning piglets. Rajendran et al. (2013) reported Zinc (Zn) NPs to reduce somatic cell counts and increase milk production in cows. Najafzadeh et al. (2013) reported zinc oxide nanoparticles toxicity in lambs which showed liver toxicity and renal damage, and in case of sheep, it showed toxicity and damaged liver, kidney, pancreas, rumen, abomasum, small intestine, and adrenal gland (Allen et al., 1983). Furthermore, Han et al. (2016) reported ZnO NPs toxicity in testicular cells of mice and suggested the safer use of such NPs for human health. Qin et al. (2019) reported cerium oxide nanoparticles (CeO2 NPs), which showed male reproductive toxicity in mice, and could be possible in humans. However, potentialities of biogenic plant-mediated copper, and its oxide and iron, and its oxide nanoparticles could be beneficial for the animal production and health; this fact cannot be ruled out (Singh, 2019b,c).

12.7 Polymeric nanoparticles Polymeric nanoparticles are widely used in biomedical applications, as shown in Fig. 12.6. Yang and Alexandridis (2000) reported polymeric NPs as a drug carrier depending upon the biocompatibility, targeting, degradation, and controlled release kinetics. Jain et al. (2015c) reported alginate nanoparticles with lipopolysaccharide antigen for mucosal vaccination against Klebsiella pneumoniae. Nagatomo et al. (2015) reported Cholesteryl pullulan encapsulated tumor necrosis factor-nanoparticles adjuvant against the influenza virus. Cevher et al. (2015) reported chitosan-pullulan composite nanoparticles for nasal delivery of vaccines to treat and prevent diphtheria. Badiee et al. (2013) reported liposome-based particles with immunostimulatory adjuvants as antileishmanial vaccines. Leleux and Roy (2013) reported polymer-based particle carriers to modulate antigen, adjuvant delivery, and processing. Mohamed et al. (2018) reported an inactivated avian influenza vaccine using chitosan nanoparticles. Esposito et al. (2018) reported nanogel to heal wound using hyaluronic acid and retinyl palmitate. Kole et al. (2018) reported a nanoconjugated DNA vaccine using chitosan nanoparticles. Margaroni et al. (2017) reported PLGA-sLiAg-MPLA vaccine against visceral leishmaniasis using soluble Leishmania infantum antigens (sLiAg) and monophosphoryl lipid A (MPLA), a TLR4 ligand. Kang et al. (2017) reported AuNP-siRNA-glycol chitosan-taurocholic acid NPs with Akt2 siRNA to treat colorectal liver metastases. Karabasz et al. (2018) reported poly-L-lysine and poly-L-glutamic acid nanocapsules with polyelectrolyte to treat Hepatitis B virus (HBV) infection. Wang et al. (2010) reported HBsAg prophylactic vaccine carriers against HBV. Aflatoxins pose serious health, environmental, and economic problems in animal production when consumed by animals in their food. Mekawey and El-Metwally (2019) reported chitosan nanoparticles with phenolic compounds by nanoencapsulation to check the growth and production of aflatoxin production. Gu et al. (2019) reported Angelica sinensis polysaccharide (ASP) encapsulated Poly (lactic-co-glycolic acid) (PLGA) nanoparticles against infectious diseases.

FIGURE 12.6 Shows potential applications of polymeric nanoparticles.

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Udayangani et al. (2017) reported chitosan silver nanocomposites diet for fish gut immunity. Khademi et al. (2018) reported tuberculosis (TB) vaccine, which is polymers-based as a future vaccine in animal models using PLGA and chitosan polymers. Nagao et al. (2016b) reported CO-HbV to treat severe acute pancreatitis to reduce inflammation and acts as an antioxidant to protect from multiorgan damage. Baldissera et al. (2017) reported nerolidol-loaded nanospheres to treat memory impairment due to Trypanosoma evansi in mice and suggested a useful strategy to treat memory dysfunction and oxidative stress caused by T. evansi infection. Graphene represents a monolayer or a few layers of carbon atoms with a honeycomb lattice structure used as in tissue engineering, as well as for drug delivery, anticancer agent, and antibacterial. Ghorbani et al. (2019) reported mussel-inspired polydopamine (PDA) nanospheres for the application in tissue engineering. Changwa et al. (2018) reported the toxigenic fungi consisting of mycotoxins such as aflatoxins (AFs), fumonisin B, ochratoxin A, citrinin, zearalenone, zearalenol, deoxynivalenol. They are a very toxic natural occurrence of the cold climate in dairy cattle feeds. Hsu et al. (2015) reported self-assembling peptide nanofibers showed superior hemostatic activity to treat wounds using gelatin sponges, i.e., effective hemostatic bandage. Jung et al. (2015) reported gene therapy of pancreatic cancer targeting small interfering RNA (siRNA) delivery using a single-chain variable fragment targeted to human CD44 variant six conjugated to PEG-poly-L-lysine for gene therapy of pancreatic cancer. Lyer et al. (2015) reported nanotoxicology and nanosafety using a magnetic drug target for the treatment of cancer. Jaworski et al. (2019) reported pristine graphene platelets (GPs) using U87 tumors cultured on chicken embryo and HS-5 cell lines (control) and suggested an increased level of apoptotic and necrotic markers in GPs-treated cell line. Sadati et al. (2018) reported inactivated influenza virus vaccines with CpG and Chitosan coating for humoral and cellular immunities in the animal model. Kalantari et al. (2019) reported Livergol (silibinin, silidianin, isosilibinin, silicristin, and flavonolignan) derived from Silybummarianum (milk thistle) as polymeric forms showed hepatoprotective agent to treat and protect hepatitis, cirrhosis, and toxicants.

12.8 Conclusions Nanotechnology (NT) is providing nano-drug and delivery systems using liposomes NPs, polymeric NPs, dendrimers NPs, metal NPs, etc. Liposomes are modifiable and micellar nanoparticles, which have loading capacity and stability; dendrimers are functionalized materials, with immense benefits in animal production; polymeric nanoparticles are stable with biological fluids; Metallic NPs have unique sizes, shapes, diagnostic, and therapeutic properties. These unique properties of all the above described NPs in this book chapter offer them a variety of biomedical applications for animals (domestic or veterinary animals) like biological markers detection, imaging, drug delivery, diagnostics, and therapeutics. Targeted deliveries of drugs for infections in animals provide both short-term and long-term treatment strategies. NT has revolutionized animal production and health using modern tools for diagnostics and therapeutics applications. Nanomicrofluidics, nanomaterials, and nanobiosensors can solve the problems related to animal production and their health monitoring. Nanodevices based on different NPs offer a significant role in animal production and health; these NPs can also be used for promoting animal growth, as antimicrobials, and for nutrient delivery. Thus, animal production and health are the backbone of the economy for any country. Due to the increasing demand for animal production in the livestock industry, the use of antibiotics to increase yield is being practiced, and longer use of antibiotics will cause microbial antibiotic resistance, which will pose a severe problem not only in animal production but also their health. Hence, utilization of these NPs will help to solve all the problems pertaining to animal production and health, because of the reason that these NPs have enormous applications, which are elaborated above in this chapter. There are many modes of mechanism data of these NPs toward disease treatment that needs to be explored. Thus, a study in that direction needs to be carried out.

Acknowledgment All the authors are thankful to Indira Gandhi National Tribal University, Amarkantak, M.P., India, for providing facilities to prepare this book chapter.

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Singh, R.P., 2016. Nanobiosensors: potentiality towards bioanalysis. J. Bioanal. Biomed. 8, e143. https://doi.org/10.4172/1948-593X.1000e143. Singh, R.P., 2017. Application of nanomaterials towards development of nanobiosensors and their utility in agriculture. In: Prasad, R., Kumar, M., Kumar, V. (Eds.), Nanotechnology: An Agricultural Paradigm. Springer Publisher, New York, USA, pp. 293e303. Ch 14. Singh, R.P., 2019a. Chap 11. In: Singh, R.L., Mondal, S.K. (Eds.), Utility of Nanomaterials in Food Safety. Food Safety and Human Health. 2019 Elsevier Inc. ISSN: 2523-8027. Singh, R.P., 2019b. Potential of biogenic plant-mediated iron and iron oxide nanostructured nanoparticles and their utility. In: Prasad, R. (Ed.), Nanotechnology in the Life Sciences: Plant Nanobionics, Volume. 2, Approaches in Nanoparticles Biosynthesis and Toxicity. Springer Nature, Switzerland AG. Chap. 04. Singh, R.P., 2019c. Potentialities of biogenic plant-mediated copper and copper oxide nanostructured nanoparticles and their utility. In: Prasad, R. (Ed.), Nanotechnology in the Life Sciences: Plant Nanobionics, Volume 2, Approaches in Nanoparticles Biosynthesis and Toxicity. Springer Nature, Switzerland AG. Chap. 05. Singh, R.P., Pandey, A.C., 2011. Silver nano-Sieve using 1, 2-benzenedicarboxylic acid as a sensor for detecting hydrogen peroxide. Anal. Methods 3, 586e592. Royal Society of Chemistry Publishing, Cambridge (UK). Singh, R.P., Kang, D.Y., Choi, J.W., 2010. Electrochemical DNA biosensor for the detection of sanguinarine in adulterated mustard oil. Adv. Mater. Lett. 1 (1), 48e54. Singh, R.P., Kang, D.Y., Oh, B.K., Choi, J.W., 2009a. Polyaniline based catalase biosensor for the detection of hydrogen peroxide and azide. Biotechnol. Bioproc. Eng. 14 (4), 443e449. Singh, R.P., Kim, Y.J., Oh, B.K., Choi, J.W., 2009b. Glutathione-s-transferase based electrochemical biosensor for the detection of captan. Electrochem. Commun. 11, 181e185. Singh, R.P., Oh, B.K., Koo, K.K., Jyoung, J.Y., Jeong, S., Choi, J.W., 2008. Biosensor arrays for environmental pollutants detection. BioChip J. (BCJ) 2 (No. 4), 223e234. Singh, R.P., Kang, D.Y., Choi, J.W., 2011a. Nanofabrication of bio-self assembled monolayer and its electrochemical property for toxicant detection. J. Nanosci. Nanotechnol. 11, 408e412 (Publisher: American Scientific Publishers.). Singh, R.P., Kumar, K., Rai, R., Tiwari, A., Choi, J.W., Pandey, A.C., 2012a. Synthesis, characterization of metal oxide-based nanomaterials and its application in biosensing. International. In: Rai, R. (Ed.), Synthesis, Characterization and Application of Smart Material. Nova Science Publishers, Inc USA, pp. 225e238 (Chapter 11). Singh, R.P., Choi, J.W., Tiwari, A., Pandey, A.C., 2012b. Utility and potential application of nanomaterials in medicine. In: Tiwari, A., Ramalingam, M., Kobayashi, H., Turner, A.P.F. (Eds.), Biomedical Materials and Diagnostic Devices. John Wiley & Sons, Inc., Hoboken, NJ, USA https://doi.org/ 10.1002/9781118523025 ch7. Smekalova, M., Aragon, V., Panacek, A., Prucek, R., Zboril, R., Kvitek, L., 2016. Enhanced antibacterial effect of antibiotics in combination with silver nanoparticles against animal pathogens. Vet. J. 209, 174e179. Stanisavljevic, M., Chomoucka, J., Dostalova, S., Krizkova, S., Vaculovicova, M., Adam, V., Kizek, R., 2014. Interactions between CdTe quantum dots and DNA revealed by capillary electrophoresis with laser-induced fluorescence detection. Electrophoresis 35, 2587e2592. Stankic, S., Suman, S., Haque, F., Vidic, J., 2016. Pure and multi metal oxide nanoparticles: synthesis, antibacterial and cytotoxic properties. J. Nanobiotechnol. 14, 73. Tada, R., Suzuki, H., Takahashi, S., Negishi, Y., Kiyono, H., Kunisawa, J., Aramaki, Y., 2018. Nasal vaccination with pneumococcal surface protein A in combination with cationic liposomes consisting of DOTAP and DC-chol confers antigen-mediated protective immunity against Streptococcus pneumoniae infections in mice. Int. Immunopharm. 61, 385e393. Tao, Y., Zhang, Y., Ju, E., Ren, J., Qu, X., 2015. One-step synthesized immunostimulatory oligonucleotides-functionalized quantum dots for simultaneous enhanced immunogenicity and cell imaging. Colloids Surf. B Biointerfaces 126, 585e589. Taylor, R., Hare, J.P., Abdul-Sada, A.K., Kroto, H.W., 1990. Isolation, separation and characterisation of the fullerenes C60 and C70: the third form of carbon. J. Chem. Soc. Chem. Commun. 1423. Taylor, W.R.J., Hanson, J., Turner, G.D.H., White, N.J., Dondorp, A.M., 2012. Respiratory manifestations of malaria. Chest 142, 492e505. Udayangani, R.M.C., Dananjaya, S.H.S., Nikapitiya, C., Heo, G.-J., Lee, J., De Zoysa, M., 2017. Metagenomics analysis of gut microbiota and immune modulation in zebrafish (Danio rerio) fed chitosan silver nanocomposites. Fish Shellfish Immunol. 66, 173e184. Vacas-Cordoba, E., Galan, M., de la Mata, F.J., Gomez, R., Pion, M., Muñoz-Fernández, M.Á., 2014. Enhanced activity of carbosilane dendrimers against HIV when combined with reverse transcriptase inhibitor drugs: searching for more potent microbicides. Int. J. Nanomed. 9, 3591e3600. Wang, X., Dai, Y., Zhao, S., Tang, J., Li, H., Xing, Y., Qu, G., Li, X., Dai, J., Zhu, Y., Zhang, X., 2014. PAMAM-lys, a novel vaccine delivery vector, enhances the protective effects of the SjC23 DNA vaccine against schistosoma japonicum infection. PLoS One 9, e86578. Wang, X.Y., Zhang, X.X., Yao, X., Jiang, J.H., Xie, Y.H., Yuan, Z.H., Wen, Y.M., 2010. Serum HBeAg sero-conversion correlated with decrease of HBsAg and HBV DNA in chronic hepatitis B patients treated with a therapeutic vaccine. Vaccine 28, 8169e8174. Wang, X., Dang, E., Gao, J., Guo, S., Li, Z., 2013. Development of a gold nanoparticle-based oligonucleotide microarray for simultaneous detection of seven swine viruses. J. Virol. Methods 191, 9e15. Weldon, B.A., Griffith, W.C., Workman, T., Scoville, D.K., Kavanagh, T.J., Faustman, E.M., 2018. In vitro to in vivo benchmark dose comparisons to inform risk assessment of quantum dot nanomaterials. Wiley Interdiscip. Rev. Nanomed. Nanobiotechnol. 10, e1507. Yandrapu, S.K., Kanujia, P., Chalasani, K.B., Mangamoori, L., Kolapalli, R.V., Chauhan, A., 2013. Development and optimization of thiolated dendrimer as a viable mucoadhesive excipient for the controlled drug delivery: an acyclovir model formulation. Nanomed. Nanotechnol. Biol. Med. 9, 514e522.

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Yang, L., Alexandridis, P., 2000. Physicochemical aspects of drug delivery and release from polymer-based colloids. Curr. Opin. Colloid Interface Sci. 5, 132e143. Ye, S., Shao, K., Li, Z., Guo, N., Zuo, Y., Li, Q., Lu, Z., Chen, L., He, Q., Han, H., 2015. Antiviral activity of graphene oxide: how sharp edged structure and charge matter. ACS Appl. Mater. Interfaces 7, 21571e21579. Yuan, Y.-G., Peng, Q.L., Gurunathan, S., 2017. Effects of silver nanoparticles on multiple drug-resistant strains of Staphylococcus aureus and Pseudomonas aeruginosa from mastitis-infected goats: an alternative approach for antimicrobial therapy. Int. J. Mol. Sci. 18, 569. Zavareh, S., Zarei, M., Darvishi, F., Azizi, H., 2015. As (III) adsorption and antimicrobial properties of Cuechitosan/alumina nanocomposite. Chem. Eng. J. 273, 610e621. Zhang, X.F., Liu, Z.G., Shen, W., Gurunathan, S., 2016. Silver nanoparticles: synthesis, characterization, properties, applications, and therapeutic approaches. Int. J. Mol. Sci. 17, 1534.

Further reading Colley, D.G., Bustinduy, A.L., Secor, W.E., King, C.H., 2014. Human schistosomiasis. Lancet 383, 2253e2264. Gurunathan, S., Woong Han, J., Kim, J., 2013. Green chemistry approach for the synthesis of biocompatible graphene. Int. J. Nanomed. 2719. Hassan, A.R., De la Escosura-Muñiz, A., Merkoçi, A., 2015. Highly sensitive and rapid determination of Escherichia coli O157:H7 in minced beef and water using electrocatalytic gold nanoparticle tags. Biosens. Bioelectron. 67, 511e515. Ionov, M., Ciepluch, K., Klajnert, B., Glinska, S., Gomez-Ramirez, R., de la Mata, F.J., Munoz-Fernandez, M.A., Bryszewska, M., 2013. Complexation of HIV derived peptides with carbosilane dendrimers. Colloids Surf. B Biointerfaces 101, 236e242. Kumar, R., Singh, R.K., Moshkalev, S.A., 2019. Graphene/graphene oxide and carbon nanotube based sensors for the determination and removal of bisphenols. In: A New Generation Material Graphene: Applications in Water Technology. Springer International Publishing, Cham, pp. 329e372. Lawlor, C., O’Connor, G., O’Leary, S., Gallagher, P.J., Cryan, S.-A., Keane, J., O’Sullivan, M.P., 2016. Treatment of Mycobacterium tuberculosisinfected macrophages with poly(lactic-Co-glycolic acid) microparticles drives NFkB and autophagy dependent bacillary killing. PLoS One 11, e0149167. Sakushima, K., Hayashino, Y., Kawaguchi, T., Jackson, J.L., Fukuhara, S., 2011. Diagnostic accuracy of cerebrospinal fluid lactate for differentiating bacterial meningitis from aseptic meningitis: a meta-analysis. J. Infect. 62, 255e262. Singh, R.P., Shukla, V.K., Yadav, R.S., Sharma, P.K., Singh, P.K., Pandey, A.C., 2011b. Biological approach of zinc oxide nanoparticles formation and its characterization. Adv. Mater. Lett. 2 (4), 313e317. Teles, R.H.G., Moralles, H.F., Cominetti, M.R., 2018. Global trends in nanomedicine research on triple negative breast cancer: a bibliometric analysis. Int. J. Nanomed. 13, 2321e2336. Volkov, Y., McIntyre, J., Prina-Mello, A., 2017. Graphene toxicity as a double-edged sword of risks and exploitable opportunities: a critical analysis of the most recent trends and developments. 2D Mater. 4 (2). Art. no. 022001. Zeng, L., Li, J., Li, J., Zhang, Q., Qian, C., Wu, W., Lin, Z., Liang, J., Chen, Y., Huang, K., 2015. Effective Suppression of the kirsten rat sarcoma viral oncogene in pancreatic tumor cells via targeted small interfering RNA delivery using nanoparticles. Pancreas 44, 250e259. Zhou, J., Neff, C.P., Liu, X., Zhang, J., Li, H., Smith, D.D., Swiderski, P., Aboellail, T., Huang, Y., Du, Q., Liang, Z., Peng, L., Akkina, R., Rossi, J.J., 2011. Systemic administration of combinatorial dsiRNAs via nanoparticles efficiently Suppresses HIV-1 infection in humanized mice. Mol. Ther. 19, 2228e2238.

Chapter 13

Cell signaling and apoptosis in animals M. Naveen Kumar1, Shivaleela Biradar2 and R.L. Babu2 1

Department of Biotechnology and Genetics, M.S. Ramaiah College of Arts, Science and Commerce, Bengaluru, Karnataka, India; 2Department of

Bioinformatics and Biotechnology, Karnataka State Akkamahadevi Women’s University, Jnanashakthi Campus, Vijayapura, Karnataka, India

13.1 Introduction Cell signaling and apoptosis are very important aspects in the operation of cellular system. Cell-cell communication is due to chemical signaling for the purpose of development, tissue repair, and immunity, as well as normal tissue homeostasis. If there is any defect in interaction of cell signaling, it leads to diseases such as autoimmunity, diabetes, and cancer (Vlahopoulos et al., 2015; Wang et al., 2013). Apoptosis is programmed cell death, which occurs in multicellular organisms and happens when cells deliberately decide to die for the maintenance of cell population in tissues in some pathological conditions and during the development of organisms (Leist and Jaattela, 2001).

13.2 Cell signaling in animals During 1855, Claude Bernard invented the concept of cellular signaling. He explained that some internal secretions of ductless glands release into the bloodstream and hold an effect on distant cells. In a similar concept, in 1880, Charles Darwin and his son Francis Darwin demonstrated the phototropism of coleoptiles in plants. They described certain influence is transferring from tip to more basal areas of shoot for inducing the curvature and regulating the growth of a plant (Bennett, 1998). Later, this messenger or transmittable factor was coined as auxin. While in the case of animals, the receptor was discovered by John Langley and his student Thomas Elliott (Langley, 1901) while studying the sympathetic neuro-effector transmission. In early 1905, Ernest Starling termed the word “hormone” to describe the chemical messengers that travel from cell to cell along with bloodstream and it may coordinate the activities and growth of various organs in the body (Starling, 1914). During the 1950s the downstream intracellular events started unfolding based on the discoveries of chemical messengers and receptors. Cells are the basic structural, functional, and biological elements of any organism. All cells are communicating with each other by defined manner, which we call as cell signaling or signal transaction. The signals involve chemical signals from other cells or from an extracellular fluid environment, and these cells will also receive signals from environments, such as the digestive and respiratory system. The signal transduction involved five sequential steps; first, signaling molecule that contains the chemical signal; second, signal receptor-receiver of signals; third, signal conveyer (secondary messenger); fourth, signal transducer that amplifies the signal; fifth, signal that generates a change in the function of cells (Starling, 1914).

13.3 Classification of cell signaling in animal cells In multicellular organisms, most of the signals are chemical in nature, which are hormones, growth factors, neurotransmitters and components of extracellular matrix. The effect of these molecules is exerted locally, or they may transmit over long distances. The cell signaling classification is mainly based on the distance traveled by the signal within the organism. The classified signals are Autocrine, Paracrine, Endocrine, and Signaling by direct contact.

13.3.1 Autocrine Autocrine is a type of cell signaling, and it secretes autocrine agents to bind its autocrine receptors. The autocrine agents maybe hormones or chemical messengers that bind to the receptor of the same cell to make changes in the cell; autocrine signals plays a pivotal role in the development of the cell and in the immune response (Pandit, 2007) (Fig. 13.1).

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13.3.2 Paracrine In paracrine signaling, the cells are communicating with nearby cells through signaling molecules. For instance, if cell synthesized proteins they can diffuse over relatively shorter distance leads to change in the function in nearby cells, this phenomenon is called as paracrine interaction, and diffusible proteins are called as paracrine factors or growth and differentiation factors(GDFs) (Gilbert, 2006). Paracrine signaling molecules are intrinsically unstable or degradable, and thereby it is having limitations to travel all over the body (Fig. 13.2). A unique instant of paracrine signaling is synaptic signaling, where neurotransmitters are signaling molecules with small range, and are moving between neurons and between the neurons and muscle cells.

13.3.3 Endocrine Endocrine derived from the Greek word Endon means “within,” and kinesin means “to release.” In this type of cell signaling, the specialized cells released the hormones into the extracellular medium like blood stream or lymphatic fluids in animals and carried out to reach distant target cells throughout the body (Fig. 13.3). If one part of the body produces signals and signals is riding through circulatory system to another part of the body,endocrine glands in humans such as pituitary, thyroid, hypothalamus, pancreas, and gonads, release the various hormones in regulating development and physiology (Marieb, 2014).

13.3.4 Signaling through direct contact In animals, the signaling through direct contact is by gap junction (Fig. 13.4). Gap junctions are accumulation of intracellular channels. These channels allow small signaling molecules, which are called intracellular mediators and are scattered between 2 cells by the direct connection of cytoplasm of 2 cells. Small molecules such as ions, amino acids, nucleotides, secondary messengers, small metabolites, and electrical impulses are exchanged between two adjacent cells

FIGURE 13.1 Autocrine signaling.

FIGURE 13.2 Paracrine signaling.

FIGURE 13.3 Endocrine signaling.

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(Kelsell et al., 2001; White and Paul, 1999). Gap junctions are composed of proteins encoded by gene family “connection,” and the gap junction channels are composed of two connections, which joins the intracellular space (Willecke et al., 2002). Basically, gap junction occurs in all tissues of the body except fully developed muscle of adults and mobile cells like erythrocytes or sperms (Goodenough and Paul, 2009; Lampe and Lau, 2000, 2004; Maeda et al., 2009; Mese et al., 2007). A special case of direct signaling is by complementary proteins on the surface of 2 cells that bind to each other. After binding, there is a change in the conformation of one or both proteins and starts the transmission of signals. This type of signaling is very important in immune system, the immune cells uses the cell surfaces markers to recognize cell is that is self cell and the cell that is infected by pathogens.

13.4 Signaling receptors In multicellular organisms, specific receptor protein binds to specific signaling molecules and begins the physiological response. The receptor binds to its signaling molecule as exactly the enzyme binds to its substrate. For example, insulin receptor binds to insulin, dopamine receptor binds to dopamine, nerve growth factor receptor binds to nerve growth factor, etc. In cells, a million types of receptors exist, and distinct cell types have distinct groups of receptors. Mainly there are two types of signaling receptors are present such as Intracellular signaling receptor and Cell-surface receptors.

13.4.1 Intracellular signaling receptors The intracellular signaling receptor is also known as cytoplasmic receptors or internal receptors. These receptors are found in the cytoplasm, which gives a response to hydrophobic ligands that can easily travel in the cytoplasm. Once these molecules enter the cell, they bind to proteins that mediate the gene expression. However, these ligands act as regulators of the transcription. When signaling molecules (ligand) binds to its receptor protein in the cytoplasm, the protein changes its conformation and reveals the DNA binding site on the protein. These two complexes pass the nucleus and bind to the specific site of DNA to initiate the transcription. Transcription is a process where the information of DNA is transferring through mRNA (messenger RNA) and translating into protein. Intracellular receptors directly affect gene expression excluding to pass the signal to other receptors. Nuclear receptors are one of the important families of intracellular receptors and include receptors for thyroid hormone, steroid hormone, vitamin D, retinoid, etc, Interestingly, the structures of all nuclear receptors are same, and the ligand structure is different (Mary et al., 2018).

13.4.2 Cell- surface signaling receptors Cell surface receptors are also known as membrane receptors or transmembrane receptors. In all eukaryotes a huge set of genes encodes for proteins that function on the cell membrane as cell surface receptors. Membrane receptor families are distinctly based on their ability to identifying the ligands, the biological responses they produce, and newly according to their primary structure. Cell surface receptor activity is regulated by diverse ligands. Cell surface receptors are lipids, carbohydrates, proteins, peptides, and small organic molecules (Lemmon and Schlessinger, 2010). These are special integral membrane proteins and communicate between cell and extracellular space. The ligands may be neurotransmitters, hormones, cytokines, cell adhesion molecules, nutrients, and growth factors. These ligands bind to cell surface receptors to produce a change in cell activity and metabolism. Types of cell-surface receptors: Basically, three types of cell-surface receptors viz G-protein coupled receptors, ion channel-linked receptors, and enzyme-linked receptors.

FIGURE 13.4 Signaling through direct contact.

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13.4.2.1 G-protein-coupled signaling receptor (GPCR) The G-protein-coupled signaling receptor is also known as seven-transmembrane domain receptor. In eukaryotes, the GPCRs are huge and mostly various sets of membrane receptors; they transmit the signals to intracellular targets by the intermediator guanine nucleotide-binding proteins called G-proteins. GPCRs are encoded by nearly 800 different human genes for diverse extracellular ligands, such as hormones, chemokines, neurotransmitters, calcium ions, and sensory stimuli (Bjarnadottir et al., 2006). Additionally, the GPCRs receptors are target for w30% of all the drugs available in market (Hopkins and Groom, 2002). GPCRs consisting of seven-transmembrane a-helices are connected by loops differing in length. In vertebrates, the GPCRs are usually divided into five types based on their sequence and structural similarity, which are rhodopsin (family A), secretin (family B), glutamate (family C), adhesion, and Frizzled or Taste2 (Fredriksson et al., 2003). GPCRs mediated cell signaling pathway involves the association of other growth factors as a signaling molecule. When the growth factor interacts with GPCRs it conveys the signal inside the cell to produce necessary proteins for cell division, cell proliferation, and cell growth. When these signaling molecules bind to the GPCRs, the cytosolic domains get conformational changes and get activated. Further, activated GPCRs interacts with G-protein; G-protein is a heterotrimeric membrane-anchored protein, which has three structural subunits as a, b, and g domain, in which a and g are membrane-anchored domains. The activated G-protein substitutes GTP from GDP with a subunit and the G-protein subunits disassociated as a subunit and bg subunits separately. The activated a-GTP complex further activates adenylyl cyclase that converts ATP into cAMP. The cyclic AMP is a signal conveyer and also called as a secondary messenger activates the multiple downstream signal transducers. One such is protein kinase A (PKA) which has multiple structural units as regulatory units and catalytic units. Then the cAMP is attached to the regulatory unit that deactivates the regulatory unit by changing its structure that allows the enzymatic unit free. PKA enzymatic unit phosphorylates other downstream molecules. Finally, they activate the transcription factor CREB (cAMP response element-binding protein), and later it enters the nucleus and binds to the response element in the gene that allows further production of downstream proteins for cell growth (Pierce et al., 2002; Rosenbaum et al., 2009; Strader et al., 1994).

13.4.2.2 Ligand-gated ion channels The ligand-gated ion channels are members of a wide family of homologous, multipass transmembrane proteins. Ligandgated ion channels are also known as ion channel-linked receptors or ionotropic receptors. When a ligand binds to these receptors, they accelerate to open the channels that are present in the cell membrane and those channels permitting some specific ions to pass through. Cell-surface receptors have a large membrane-spanning region to build channels. Certain receptors in the central nervous system play a similar function as ligand-gated channels, including GABAA, glycine, 5HT3, and nicotinic acetylcholine (nACh). In the membrane-spanning region, several amino acids are hydrophobic in nature. Inversely, an amino acid that lies inside the channel is hydrophilic to permit the flow of ions or water. When a ligand binds to the channel’s extracellular area, there is aconformational change in the protein structure that enables ions like sodium, potassium, calcium, magnesium, and hydrogen to go through. For example, the neurotransmitter binds to the ion channel receptor to open the channel for passing the synaptic signals between electrically excitable cells (Uings and Farrow, 2000; Unwin, 1993).

13.4.2.3 Enzyme-linked receptors Enzyme-linked receptors are also known as catalytic receptors. These are the cell surface receptor and the binding of a ligand to extracellular domains resulting in enzymatic activity on the intracellular domains. Usually, the enzyme-linked receptors have extensive extracellular and intracellular domains, although the transmembrane region includes a single alpha-helical structure of the peptide strand. Once the ligand binds to the extracellular domain the signal travels over the membrane and activates the enzyme. This activated enzyme triggers a chain of cellular responses.

13.4.2.4 Receptor tyrosine kinases (RTKs) Receptor tyrosine kinase is example of an enzyme-linked receptor. When a proper signal binds to the receptor tyrosine kinase, the internal tyrosine kinase activity promotes the autophosphorylation of tyrosine inside the receptor. There is a large array of intracellular proteins that binds to the phosphotyrosine residues; these proteins are having sequence similarity and are called SH2protein domains. The SH2domain protein intercommunicates with Sos (son of sevenless) protein. The Sos protein triggers Ras (rat sarcoma) protein by inducing Ras protein to bind GTP. Ras is a monomeric G protein that is encoded by ras protooncogene. The stimulated Ras protein activates Raf protein kinase. This activated Raf protein kinase stimulates the mitogen-activated protein kinase (MAP kinase) through the covalent phosphorylation of tyrosine and threonine. The stimulated MAP kinase walks into the nucleus from the cytoplasm. Where gene regulatory proteins phosphorylate by MAP kinase and induce the gene transcription. Examples for receptor tyrosine kinases are epidermal

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growth factor (EGF) receptor, fibroblast growth factor (FGF) receptor, platelet-derived growth factor (PDGF) receptor, vascular endothelial growth factor (VEGF) receptor, nerve growth factor (NGF) and insulin receptor (Dudek, 2007; Lemmon and Schlessinger, 2010; Uings and Farrow, 2000).

13.5 Second messengers in animal cell signaling The secondary messenger was discovered by Earl Wilbur Surtherland Jr, and he won the Nobel prize in 1971. In a cell, extracellular domains have an integral transmembrane receptor protein that binds to the specific biochemical signals, including hormones, neurotransmitters, and immunoglobulin, and therefore transfer the information through the membrane to the cytoplasm this process is called as signal transduction or transmembrane signaling, which includes number of secondary messenger signaling molecule generation. Secondary messengers are ions and small molecules, which convey the message obtained by cell surface receptor to effector proteins; popular examples of secondary messengers are calcium, cyclic nucleotide, phosphoinositides, and diacylglycerol. Secondary messengers greatly amplify the signal strength to produce a change in cell activity. For instance, activities such as phosphorylation of receptors and downstream proteins are largely influenced by second messengers (Newton et al., 2016). There are three basic types of secondary messenger molecules involved in signal transduction (Karp et al., 2009; Newton et al., 2016; Rastogi, 2006). Hydrophobic molecules: These are water-insoluble molecules, which are membrane-bounded and spread the signals from the plasma membrane receptors to target proteins inside the cell that can be reached and regulated; such molecules are diacylglycerol, and phosphatidylinositol. Diacylglycerol (DAG): Diacylglycerol impels protein kinase C activity by greatly increasing the affinity of the enzyme for calcium ions. Protein kinase C phosphorylates certain threonine and serine residues in effector proteins. The effector proteins include calmodulin, glucose transporter, cytochrome P450, HMG-CoA, and other similar molecules. Phosphatidylinositol: Phosphatidylinositol is a minor component in eukaryotic cell membrane, and it is a negatively charged phospholipid. The inositol would be phosphorylated to form Phosphatidylinositol-4-phosphate (PIP), Phosphatidylinositol-4,5-bisphosphate (PIP2), Phosphatidylinositol-3,4,5-triphosphate (PIP3). Intracellular phospholipase C (PLC) enzyme hydrolyzes PIP2, which forms diacylglycerol (DAG) and inositol-1,4,5-triphosphate (IP3). Hydrophilic molecules: These molecules are water soluble associated within the cytosol; such molecules are cAMP, IP3, cGMP, and Ca2þ. cAMP: cAMP is a second messenger synthesized from ATP by the enzyme adenylyl cyclase and is activated by a G protein and G protein is activated by hormone binding to its receptor. This leads to a specific response in the cell. PKA is a cAMP-dependent protein kinase that phosphorylates target proteins and cAMP binds to CREB (cAMP response elementbinding protein) protein, and the resultant complex regulates the transcription or expression of genes. cGMP: cGMP is another type of secondary messenger synthesized from GTP by the enzyme guanylyl cyclase. The nitric oxide triggers the synthesis of cGMP. cGMP stimulated protein kinase that includes both catalytic and regulatory subunits. Protein kinase G (PKG) mediates the impact of cGMP. Gases: These gases spread through the cytosol, as well as across the cellular membrane, viz. carbon monoxide (CO), nitric oxide (NO), and hydrogen sulfide (H2S).

13.6 Pathways of cell signaling WNT pathway: Wnt pathway is also termed as the b-catenin signaling pathway. Wnt pathway is evolutionary conserved and very important during embryonic development of all animal species. Wnt pathway involved in the regeneration of tissues and numerous other processes. Wnt gene was discovered by Roel Nusse and Harold Varmus in 1982, in the study of mouse mammary tumor virus (Klaus and Birchmeier, 2008) and the name Wnt is formed by the drosophila segment polarity gene wingless and homologous gene in vertebrate int1 or integrated (Wodarz and Nusse, 1998). Wnt pathways are identified based on the frizzled protein receptors downstream signaling. They are canonical Wnt pathway or b-catenin dependent pathway, noncanonical or b-catenin-independent pathway, which can be subdivided into planar cell polarity, and wnt/calcium pathway (Komiya and Habas, 2008). NF-kB pathway: NF-kB (nuclear factor kappa light chain enhancer of activated B cells) is a heterodimeric nuclear transcription factor binds to specific DNA conserved sequence present within the intronic immunoglobulin kappa light chain enhancer gene in mature B cells and other genes (Sen and Baltimore, 1986). Binding of NF-kB is responsible for the inducible activity of the enhancer element of the immunoglobulin gene. NF-kB is found in almost all animal cell types and is responsive to stimuli such as stress, cytokines, free radicals, ultraviolet irradiation, and bacterial, or viral antigens (Roman-Blas and Jimenez, 2006; Yamamoto and Gaynor, 2001). NF kappa B signaling pathway initiates with signaling molecule includes ROS (Reactive oxygen species), ionizing radiation, IL-1 (interleukin-1),TNF-a/b (Tumor necrosis

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factor-a/b), different foreign material e.g., LPS, Flagellin proteins (Karin, 2008; Seaman, 2002). Two major classes of NFkB proteins are present in mammalian cells, such as the class I comprises of p50/p105 (NF-kB1), p52/p100 (NF-kB2), and Class II includes c-Rel, Rel-B, and p65 (Rel-A) proteins. Both the classes of NF-kB transcription factor share structural homology containing a conserved stretch of 300 amino acids, designated as the Rel homology domain (RHD). The N-terminal DNA-binding domain (DBD) of the protein serves as a binding site for NF-kB TFs and inhibitory IkBa protein. The C-terminus of class I proteins contain ankyrin repeats and possess transrepression activity, while the C-terminus of class II proteins has transactivation domain. In normal cells NF-kB is present in the cytosol in an inactive form, and the activation of this nuclear factor is regulated by its endogenous inhibitor IkB, which complexes and sequesters NF-kB in the cytoplasm. Following stimulation, the successive activation of various kinases leads to the phosphorylation and degradation of IkB molecule in the cytoplasm and subsequent release of NF-kB, which is later translocated to the nucleus and activates the transcription of multiple genes that includes TNF-a, IL-6, IL-8, and other chemokines MHC class II, ICAM-1, iNOS, and COX-2 (Barnes and Karin, 1997). As NF-kB controls many genes involved in inflammation, it is found to be constantly active in many inflammatory diseases (Neurath et al., 1998; Schreiber et al., 1998; Seaman, 1997). Nitric oxide pathway: The discovery of nitric oxide as a signaling molecule in the cardiovascular system was made by Robert Furchgott, Louis Ignarro, and Ferid Murad, who got the Nobel prize in medicine or physiology in 1998 (Bryan et al., 2009). Nitric oxide (NO) is produced by the amino acid arginine and synthesized by the enzyme nitric oxide synthase. Nitric oxide is a signaling molecule of paracrine signalings, such as steroid hormones. NO directly diffuses through the plasma membrane of its target cell. Once the nitric oxide molecule is synthesized, it diffuses out of the cell and affects nearby cells in paracrine signaling. An example of NO signaling is the dilation of blood vessels. Nitric oxide plays a significant role in cardiovascular system in vasodilation, which decreases blood pressure and increases the oxygen level in blood. NO acts as a messenger molecule to convey the message to the blood vessels to widen or dilate. Nitric oxide (NO) is a molecular mediator of vasodilation, inflammation, immunity, thrombosis, neurotransmission, and many more physiological processes (Schmidt and Walter, 1994). NO is a gaseous signaling molecule that regulates various physiological and pathophysiological responses in the human body. The function of NO varies from a potent vasodilator, neurotransmitter, to inducer of pathogen death and tissue damage depending on the tissue (Lo et al., 2002). The mouse macrophages produce nitrite and nitrate in response to bacterial lipopolysaccharide (Stuehr and Marletta, 1985), and NO proved to be an intermediate in the process. NO was found to be an effector molecule in macrophage-mediated cytotoxicity (Hibbs et al., 1988), and high levels of NO are produced in response to inflammatory stimuli and mediate proinflammatory and destructive effects. The role of NO in the respiratory system has been studied extensively. NO appears to act as a neurotransmitter in NANC nerves, regulates smooth muscle tone, and also involved in the host defense in bronchial epithelium. It acts as an inflammatory mediator in the pathological status of lungs. In addition, NO can be used as a marker for lung inflammatory diseases (Nevin and Broadley, 2002; Ricciardolo et al., 2004). Nuclear receptor pathway: Nuclear receptors are the large classes of transcriptional regulators in animals. They regulates different functions like development, reproduction, homeostasis and metabolism (Laudet and Gronemeyer, 2002). Nuclear receptors are ligand-activated transcription factors, and ligands, including steroid hormones, thyroid hormones, retinoic acid, and vitamin D, have a similar structure and functional elements (Sever and Glass, 2013). The nuclear receptors have two major functional domains, DNA-binding domain (DBD) and ligand-binding domain (LBD). The LBD contains transactivation domains namely Activation Functional domains (AF-1 and AF-2) among which AF-2 is required for the recruitment of coregulatory elements for the transactivation process (Meneses-Morales et al., 2014). The receptors that lack the AF-2 domain does not bind to a hormone, and hence, cannot switch on ligand-dependent signal transduction pathways. The DBD region is highly conserved, situated at an amino-terminal end organized into two CyseCys zinc fingers, and the LBD is situated at the carboxy-terminal end (Evans and Hollenberg, 1988). Nuclear receptors family consists of steroid hormones chemically are lipids secreted by gonads (ovary and testis) and adrenal cortex under the influence of different hormones produced via the hypothalamic-pituitary-adrenal axis. Their lipophilic nature makes them diffuse across the plasma membrane to bind to their receptors located either in the cytoplasm as an inactive complex with heat shock proteins or on the nuclear membrane that belong to the nuclear receptor superfamily (Kicman, 2008). The genes that are regulated by nuclear receptors contain specific DNA sequences in their promoters regions, where the nuclear receptor binds. The hormone-receptor dimer activates signaling via the classical genomic pathway, nonclassical genomic pathway, and the nongenomic pathway. In the classical genomic pathway, hormone-receptor dimer binds to hormone response elements present in the promoter of the target genes and activates the transcription. The nonclassical genomic signaling includes the tethering of hormone-receptor dimer to transcription factors such as AP-1, NF-kB, or Sp1 and activates the transcription of target genes. The nongenomic signaling involves the activation of second messenger cascades, interacting with several signaling molecules like phosphatidyl-inositol 3-kinase (PI3K)/Akt, the nonreceptor tyrosine kinase c-Src (Src), Ras-Raf-1, PKA and PKC (Krishnappa et al., 2017; Ricciardolo et al., 2004). These signaling molecules, in turn, converge on

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mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase activation and activate signal transduction cascades, which are involved in deciding cellular response (Naveen Kumar et al., 2019; Parekh et al., 2000).

13.7 Computational mapping of animal cell signaling Computational mapping of cell signaling will help in understanding the pathways interaction, dynamics of signaling networks, and ligand-receptor complex binding through the computational approach (Chen and Thorner, 2005). Computational models provide the information from the published literature to produce a group of signaling elements and their interaction associated (Hughey et al., 2010). The growth of computational models examining more in-depth of cell signaling pathways at worldwide by handling distinct types of variables and resulting response will be evaluated systematically (Rangamani and Iyengar, 2008). Many biological pathway databases will provide the information regarding pathway network and signaling between the molecules such databases are KEGG (Kyoto Encyclopedia of genes and genomes), MetaCyc and EcoCyc, WIT, BioCarta, PFBP (Protein Function and Biochemical Pathways), BBID (Biological Biochemical Image Database), EMP (Enzymes and Metabolic Pathways Database), Metabolic pathway of biochemistry, and regulatory signaling pathway databases are TRANSPATH Signal Transduction Browser, SPAD (Signaling Pathway Database), CSNDB (Cell Signaling Networks Database), GeneNet, STKE (The Science Signal Transduction Knowledge Environment) connections map, GeNet (Gene Networks Database) (Wixon, 2001). Analytical models are used for studying the signal transduction and is applied in pharmacology and drug discovery to determine the ligand-receptor interactions and pharmacokinetics along with a cascade of metabolites in large networks (Eungdamrong and Iyengar, 2004). The most commonly applied strategy to make models of cell signaling is through the use of ordinary differential equation models by the term time-dependent concentration of signaling molecules as a function of other molecules downstream and upstream within the pathway (Goodenough and Paul, 2009; Poupon and Reiter, 2014).

13.8 Apoptosis Way back to 48 years the world of word “Apoptosis” raised in the scientific article to clear the concepts of death and survival of a cell in its journey of life in developing organisms is a great history today as we have ample information on the same topic with the advancement of techniques to understand much more on the behavior of science in clarifying a concept of “the programmed cell death.” Apoptosis is natural cell death in multicellular organisms and mechanistically referred to as programmed cell death. In early 1972, John F. Kerr, Andrew H. Wyllie, and A. R. Currie termed “apoptosis” to differentiate the natural death of cells from the necrosis process, which is a result of an instant injury. Apoptosis is a greek word that describes dropping off or falling off of petals from flowers or leaves from trees and the name was proposed by Professor James Cormack (Kerr et al., 1972). Apoptosis occurs in the embryonic stage, adult cells, and aging (Cotter, 2009). On an average, 60 billion cells among adult human body cells die per day, and this process of apoptosis occurs due to external signals occurring at plasma membrane receptor or otherwise by internal phenomena such as cell cycle completion, cell injury, DNA damage, oxidative stress, and chemotherapy (Cotter, 2009; Nelson, 2005). The predecided cellular signals in development of cell to adulthood un-doubtfully sacrifice several times of present cells population to provide a structural or morphological outlook in any kind of organism. During such formation, the cell-programming serving as a key player in deciding cell fate of life or dead depends on the endogenous and exogenous signals. The stringent process of cell death occurs through different mechanisms such as apoptosis, autophagy, and necrosis. However, the situation after cell death from the above three mechanisms differently affects the organism’s biological process integrity leading to several beneficial and nonbeneficial outcomes. The cell death plays a pivotal role during the development of organisms and the first to be observed in the metamorphosis of amphibians and later discovered in the developing tissues of many vertebrates and invertebrates (Clarke and Clarke, 1996). The basis of cell death was further clarified and identified by using inhibitors of transcription and translation in insects, amphibians, and rodent models, and shows the control-of-death switch in the hands of nucleic acid and protein molecules. The process of cell death can be avoided by means of substances that are released by other tissues, indicating that the signals from other cells and external factors responsibly suppress the causable cell death (Saunders, 1966). Hence, the overall progress of death and survival is programmed as predictable in the development of the organism (Lockshin and Williams, 1964). Apoptotic cells and their properties found to be similar in all animal tissue types and eventually apoptotic bodies captured inside the other cells by the process of phagocytosis, which is induced by physiological or pathological environmental stimuli by avoiding cross-inflammatory responses (Jacobson et al., 1997).

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The mechanism of the controlled cell-deletion program that is opposite to mitosis is a characteristic feature of the regulation of the animal cell population. Apoptosis is mainly involved in the turnover of cells in many healthy tissues and in the elimination of cells during normal embryonic development. Apoptosis also occurs as a defense mechanism, such as in immune reactions or when cells are damaged by disease or noxious agents (Norbury and Hickson, 2001). Exogenously it can also be facilitated by the same noxious agents in the embryo and adult animal (Kerr et al., 1972). Genetic studies in the nematode Caenorhabditis elegans set an idea of programmed cell death, and several genes involved in the death program are identified to be homologous to the mammalian genome (Hengartner and Horvitz, 1994; Yuan et al., 1993). It is noted from the study that normal cell death or it may be induced by any stimulus follows the evolutionary basis of conserved death program during the development of organisms (Jacobson et al., 1997; Weil et al., 1996).

13.9 Classification of cell death in animal cells Apoptosis-the programmed cell death typically maintains the cell populations in tissues during development and maturation as a part of the physiological mechanism. A variety of stimuli and environment easily trigger the cell stress but fails to achieve their target, due to unresponsiveness to some type of stimulus. The drugs used in chemotherapy and hormones induced results in cell death by apoptosis, and other cells are not even stimulated and unaffected by such modulators. These distinguished mechanisms occur though receptor-mediated, stimuli-mediated, and even by intracellular-mediated pathway (Pfeffer and Singh, 2018). Overall, apoptosis is a coordinated process that involves the activation of a group of composed cascades that link from initiating stimuli to the final departure of the cell (Elmore, 2007) (Fig. 13.5). After the final departure of cells as apoptotic bodies during maturation, they eventually are degraded rapidly and disappear by surrounded macrophage cells. Functionally large-scale removal of apoptotic bodies succeeded in an hour or less than leaving few dead cells in the tissue. The quantitative measurement of the clearance of dead cells by phagocytosis after apoptosis is still immeasurable completely and understudied event in death program (Arandjelovic and Ravichandran, 2015). The intracellular death-program generally follows the linkage of events by expressing protein components in all cells of a specific tissue, then execution by proteolytic cascade and controlled activation of regulatory genes and proteins. Evidentially, cycloheximide along with staurosporine inhibit the protein synthesis in many nucleated animal cells and induced programmed-cell-death among all cell types, including dissociation of the mouse embryo (Ishizaki et al., 1995; Tamaoki and Nakano, 1990), explant cultures, and other rodent organs (Weil et al., 1996). The exception in human red blood cells due to lack of nucleus and staurosporine induces apoptosis through cytoplasts in enucleated cells (Jacobson et al., 1997). Apoptosis deficient laboratory nematodes show 15% more cells and function less compared to the normal one (Ellis et al., 1991). Mutant flies die early in development due to untimely cell death program (White, 1996). Mice with mutant caspase-3 showed an excess of cells in the nervous system die parentally due to disrupted apoptosis cascade (Kuida et al., 1996). Apoptosis serves functional and morphological development of animals by sculpturing structures, removing unneeded structures, controlling cell numbers, eliminating abnormal, misplaced, nonfunctional, or harmful cells, and producing differentiated cells (Jacobson et al., 1997).

FIGURE 13.5 Classification of cell death in animal tissue/cells.

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13.9.1 Autophagy Cell death status by autophagy in any organism shows bulk lysosomal degradation of cytoplasmic material in the autophagosomes, which has a characteristic two membranes and degenerating cytosol or cytoplasmic organelles (Levine and Klionsky, 2004; Levine and Kroemer, 2008). Autophagy is a tightly regulated process involved in the turnover of proteins and whole organelles, thereby eliminating excessive or damaged organelles, sometimes specifically the target organelles such as mitochondria (mitophagy) and the endoplasmic reticulum (reticulophagy) (Rubinsztein et al., 2007; Shintani and Klionsky, 2004). The damaged or aged cells or organelles are eliminated by the constitution of two death/survival programs, i.e., apoptosis and autophagy. Generally, similar stimuli found to be induced either apoptosis or autophagy, but mixed reactions are observed in response to the common stimuli (Maiuri et al., 2007). The cells deceased with an autophagic condition show no association with phagocytes in contrast to the apoptosis pathway (phagocytosis following chromatin condensation) (Baehrecke, 2005; Clarke, 1990). The catabolic process of the autophagic pathway occurs with the formation of autolysosomes by the fusion of autophagosomes and lysosomes (Gonzalez-Polo et al., 2005; Levine and Kroemer, 2008). Further, the inner membrane and luminal content of autophagosomes are degraded by acidic lysosomal hydrolases to complete the process. Through the catabolism of macromolecules, autophagy produces metabolic substrates to meet the biological needs of cells and allow them for adaptive protein synthesis (Lum et al., 2005). The induction of autophagic conditions is achieved by blocking autophagosomes with lysosomes, and the accumulation of autophagosomes stimulates the degradation of cytoplasmic material (Klionsky et al., 2008). In vivo studies using Drosophila melanogaster salivary glands demonstrates the reduces cell death with the knockout of genes (atg) required for autophagy (Berry and Baehrecke, 2007) and conversely other studies also observed autophagy promotes cell survival, in multiple physiological and experimental settings (Baehrecke, 2002; Clarke, 1990; Neufeld and Baehrecke, 2008). The direct stimulation of autophagy by inducing Atg1 kinase is appropriate to destroy the fat and salivary gland cells in D. melanogaster (Galluzzi et al., 2008). Incidentally, Atg1-leading autophagic cell death in fat cells involves caspase-dependent mechanisms (Scott et al., 2007), and the same is not in case of salivary gland cells point-out the distinct mechanism. Autophagy may also participate in the destruction of cells depending on the response of cytoplasm; in specific cases, clarifies its deciding role in cells fate (Berry and Baehrecke, 2007; Scott et al., 2007).

13.9.2 Necrosis Necrosis in cells is an uncontrolled phenomenon, unlike apoptosis, where large numbers of cells are affected by the necrotic process. It happens with two major mechanisms, by interference with energy supply to cells and by direct damage to the cell membrane. The morphological characterizations are cell swelling (gain in cell volume), the formation of vacuoles in cytoplasm (cytoplasmic blebs), damaged mitochondria, ribosomes detachment, rupture of lysosomes, and subsequent loss of intracellular contents by the disrupted cell membrane (Kerr et al., 1972; Majno and Joris, 1995; Trump et al., 1997). The loss of cell membrane integrity eventually recruits inflammatory-mediated cells by sending chemotactic signals to the neighboring cells/tissue through the release of the cytoplasmic contents. This inflammatory response seen in necrotic cells as they are not phagocytosed by macrophages as in the case of apoptosis (Kurosaka et al., 2003; Savill and Fadok, 2000). Cell death by necrosis, autophagy, or apoptosis depends in part on the nature of the cellular death signals, the tissue type, and the physiologic developmental stage of the organ/tissue (Fiers et al., 1999; Zeiss, 2003). The mechanism of cell death by necrosis in an alternative to apoptosis cell death, where cells are passive mode of the target due to the toxic process in the degradation of cellular contents, and this is broadly referred as oncotic necrosis (Levin et al., 1999; Majno and Joris, 1995). Evidence suggests that necrosis and apoptosis represented similar morphologic expressions with biochemical status and described as the “apoptosis-necrosis continuum” however, they never follow a single cascade to achieve their target resemble them as definitionally different (Zeiss, 2003). Majorly decrease in the availability of caspases and intracellular ATP conditions may convert the ongoing apoptotic cascade into a necrotic one (Denecker et al., 2001; Leist et al., 1997) suggest the intensity and duration of the stimulus, the extent of ATP depletion and the availability of caspases are required to distinguish apoptosis from necrosis (Elmore, 2007; Zeiss, 2003). For overruling that necrosis is an accidental and uncontrolled form of cell death, there are lots of evidence showing the stimulation of necrosis by death domain receptors and toll-like receptors, particularly in the presence of caspase inhibitors with the involvement of signal transduction pathways and catabolic mechanisms. The generalized term “necroptosis” is used to indicate regulated/controlled form of (as opposed to accidental) necrosis and may represent a convenient way to distinguish between programmed and accidental forms of necrosis (Vanlangenakker et al., 2012). Although the necrotic cell death shows the involvement of several mediators, organelles, and cellular processes, it is still unclear how they are interrelated with each other. The related phenomenal elements during necrosis may include mitochondrial alterations, lysosomal changes, lipid degradation, nuclear changes, and an increase in the cytosolic concentration of calcium through the activation of noncaspase proteases (Golstein and Kroemer, 2007; Nicotera and Melino, 2004).

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The independent mode of programmed necrotic cell death by highly inflammatory “pyroptosis” and the iron-dependent “ferroptosis” mechanisms are the two emerging necrotic pathways in the cell death program. During the pathogenic infections such as Shigellaflexneri (Sansonetti et al., 2000) a strong inflammatory response is achieved with the release of IL-1b and IL-18 inflammatory cytokines from the dying cells and engages the “inflammasome” as an innate immune response for cell death execution with caspase activation is a characteristic feature of pyroptosis (Doitsh et al., 2014; Martinon et al., 2002). Ferroptosis is an iron-dependent mode of destruction of cells by excessive production of cellular reactive oxygen species (ROS) than the physiological levels (Dixon et al., 2012). The cell undergoing ferroptosis morphologically characterized by condensation and outer membrane rupture of mitochondria (Xie et al., 2016). To execute ferroptosis, the intracellular iron, its transporter transferrin, lipid-ROS, and functional lysosomes are all essential in nonapoptotic cell death modality (Hanson, 2016; Torii et al., 2016).

13.10 Cellular and biochemical feature of apoptotic cells The apoptosis process was initially defined as cells dying through the defined morphological changes and also referred to as shrinkage necrosis (Kerr, 1971; Kerr et al., 1972). The other major morphological characteristics are the changes in nuclear chromatin condensation, compact cytoplasm, nuclear blebbing, vacuoles in the cytoplasm membrane, the formation of apoptotic bodies, and eventual cellular demise without loss of membrane integrity. Apoptotic cell death is different from classical necrosis, which results in the loss of membrane integrity, leakage of cellular components leading to inflammatory response, and consecutive degradation (Granville et al., 1998). Since the process of apoptosis as a novel form of cell death, the field has exploded into a major research area (Garfield and Melino, 1997; Geske et al., 2001). By using light and electron microscopy, various morphological changes have been identified during apoptosis (Hacker, 2000). During the early process of apoptosis at the periphery of the nuclear membrane, chromatin condensation starts, forming a ring-like structure and breaks up (karyorrhexis) (Kerr et al., 1972). Electron microscopy with staining of cells/tissue can better define the subcellular changes. The histologic examination of apoptotic cells stained with hematoxylin and eosin shows single cells or small clusters of cells and appears as a round mass with dark eosinophilic cytoplasm and dense purple nuclear chromatin fragments. During the early cell detachment from the surrounding tissue, aggregation of nuclear material, chromatin condensation, and plasma membrane blebbing occur (Fischer et al., 2008). Following this, cell fragments are separated by the process of budding, and they are sealed into a separate membrane layer around the separated cellular material and form apoptotic bodies. These apoptotic bodies consist of tightly packed cellular organelles with or without fragments of nucleus (Bottone et al., 2013). Apoptotic bodies are subsequently phagocytosed by macrophages and the parenchymal cells eventually become degraded by phagolysosomes. If the membrane-bounded buddings or nuclear fragments are not phagocytosed, they will undergo a secondary necrosis mode of degradation to eliminate the cellular wastes (Elmore, 2007; Kurosaka et al., 2003; Savill and Fadok, 2000). The cellular morphological changes during apoptosis mainly lead to loss of integrity of nucleus, organelles, and cell structure. These changes reflect in the modified biochemical parameter of molecular functions and structures, which are involved in such biological events. The main biochemical changes observed in apoptosis are activation of caspases, changes in membrane structure, recognition of phagocytic cells, breakdown of nucleic acid, and proteins (Bottone et al., 2013). Caspase activation or inactivation is analyzed by enzyme binding assays; membrane damage by the expression of phosphatidylserine on the outer layer of membrane, which also helps in the recognition of dead cells by phagocytes. Degradation of nuclear DNA content into several fragments is quantified by molecular tools to estimate the status of cell survival or death.

13.11 Apoptosis: proteins and signaling pathways The mechanism of apoptosis is complex and coordinated by many signaling pathways resulting in the morphological and biochemical cellular alterations (Wong, 2011). Mainly apoptosis is mediated by extrinsic or intrinsic pathways. During mediation by several signals, the balance between survival and death responsible genes or proteins is the utmost criteria in the maintenance of physiological development and maturation of animals. The caspases, pro-apoptotic, and antiapoptotic regulators are the key agents in determining the cell to undergo apoptosis. In the normal development of animals, apoptosis occurs for maintaining homeostasis conditions by removing potentially harmful cells where they have DNA damage or are under any stress (Plati et al., 2008). In mammalian cells, the external signals trigger the death receptor pathway (extrinsic) or the mitochondrial-mediated pathway (intrinsic). The extrinsic pathway is induced upon apoptotic stimuli comprising of external signals by binding the death-inducing ligands to the cell surface receptors. In alternate pathway, DNA damage appearing due to stress, irradiation or chemicals, growth factor deprivation, or oxidative stress leads intrinsic signals to undergo apoptosis with the involvement of the mitochondria (Hacker, 2000) (Fig. 13.6).

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FIGURE 13.6 Mechanism of intrinsic and extrinsic apoptosis pathways in animal cells.

The intrinsic apoptotic pathways involve diverse array of stimuli, which are nonreceptor-mediated and produce intracellular signals to target cells within and by mitochondrial-mediated signals. The intrinsic apoptotic pathway is initiated by internal stimuli such as genetic damage, radiation, toxins, viral infections, hypoxia, high concentrations of cytosolic Ca2þ, and due to severe oxidative stress (Karp, 2008). This results in increased mitochondrial permeability and releases pro-apoptotic cytochrome-c into the cytoplasm. Stimuli cause alterations in the mitochondrial redox that results in the formation of mitochondrial permeability transition (MPT) pore and loss in the mitochondrial transmembrane potential and release of two main groups of normally sequestered pro-apoptotic proteins from the intermembrane space into the cytosol (Saelens et al., 2004). The first is cytochrome c, Smac/DIABLO, and next is serine protease HtrA2/Omi and proteins activate the caspase-dependent mitochondrial pathway (Du et al., 2000; Garrido et al., 2006). It is regulated by a group of proteins belonging to the Bcl-2 family (Danial and Korsmeyer, 2004; Tsujimoto et al., 1984), with pro-apoptotic proteins (e.g., Bax, Bak, Bad, Bcl-Xs, Bid, Bik, Bim and Hrk) act by promoting the release cytochrome-C and the antiapoptotic proteins (e.g., Bcl-2, Bcl-XL, Bcl-W, Bfl-1 and Mcl-1) regulate apoptosis by blocking the mitochondrial release of cytochrome-c. The balanced ratio of pro- and antiapoptotic proteins initiates and define the apoptosis (Reed, 1997). Subsequent caspases are activated by the release of cytoplasmic cytochrome-c via the formation of apoptosome (cytochrome c, Apaf-1 and caspase 9) (Kroemer et al., 2007). The activated caspase 3/7 dissociates the binding of DNase inhibitor with DNase and activated DNase cleave/degrade the nuclear content. The nuclear fragments further phagocytosed by macrophages as final destination of macromolecules in deciding cell status (Wong, 2011). The binding of death ligands to a death receptor initiates the extrinsic death receptor pathway. Tumor necrosis factor receptor-TNFR family (TNFRI, TNFR2, lymphotoxin-I3R, NGFR (p75), CD40, CD27 and CD30, DR-3 (death-receptor 3) and its related protein Fas (APO-I, CD95) and their ligands are called TNF and Fas ligands (FasL) are well-known death receptors (Hengartner, 2001). The TNFR family members share similar cysteine-rich extracellular domains and have a cytoplasmic-domain of about 80 amino acids called as death domain (Ashkenazi and Dixit, 1998). This death domain plays

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a pivotal role in transferring the death signal from the surface of the cell to the intracellular signaling pathways (RubioMoscardo et al., 2005; Zhang and Fang, 2005). The intracellular death domain of death receptor recruits the adapter proteins such as TNF receptor-associated death domain (TRADD) and Fas-associated death domain (FADD), along with cysteine proteases like caspase-8 (Kroemer et al., 2007; Wajant, 2002). The ligand-receptor-adaptor protein complex known as the death-inducing signaling complex (DISC) is formed by binding of the death ligand to the death receptor, which, in turn, forms a binding site adapter (O’Brien and Kirby, 2008). Once these ligand binding, cytoplasmic adapter proteins are recruited, which exhibit corresponding death domains that bind with the receptors. Further, the complex initiates the assembly and activation of pro-caspase eight, and the activated initiator caspase eight triggers apoptosis by cleaving other executioner caspases (formation of DISC resulting in the auto-catalytic activation of procaspase-8) (Karp, 2008). The initiation, mediation, execution, and regulation of apoptosis have been primarily understood by several studies, including a genetic study conducted in C. elegans. The exploration of the molecular mechanism showed involvement of various genes such as the binding of caspase homologue CED-3, and Apaf-1 homologue CED-4 activates CED-3 to induce apoptosis. Antiapoptotic Bcl-2 homologue CED-9 is associated and makes CED-4 as inactive in normal cells. The BH3 only member homologue EGL-1 protein binds to CED-9 to displace an activate CED-4 in turn to stimulate CED-3 leading to apoptosis induction. Hence, the activation and regulation of apoptosis in eukaryotes depend on analogue to exert their effect (Jin and El-Deiry, 2005; Liu and Hengartner, 1999). Tumor suppressor proteins like p53 regulate apoptosis from initial signaling stimulation and contribute to tumor cell survival or death. The transcription factor p53 regulates the expression of Bcl-2 family proteins and showed induced apoptosis in leukemia cells (Yonish-Rouach et al., 1991). In addition to control of antiapoptotic Bcl-2 family members the p53 regulated the expression of pro-apoptotic Bax in both in vitro and in vivo systems clarify its role of guardian of the genome and as key apoptosis-regulating protein in the cell system (Cotter, 2009; Laptenko and Prives, 2006). Antiapoptotic family member Bcl-2 involved in the regulation of apoptosis is the primarily characterized and identified gene in B Cell Lymphoma. Among Bcl-2 families, the proteins such as Bcl-Xl (also known as BIM/Bcl2l1) (Boise et al., 1993), Bcl-W (Gibson et al., 1996) and Mcl1 (Kozopas et al., 1993) contain three or four Bcl-2 homologous (BH) domains required for their antiapoptotic functions. Binding studies showed that the capacity for different BH3-only proteins to bind antiapoptotic proteins is not equal and reported that distinct Bcl-2 antiapoptotic proteins could only be able to bind and restrain Bax also and not in the case of Bak (Kerr et al., 1994). The pro-apoptotic members Bax and Bak can form pores (MPT) or interacts with pore-forming proteins at the level of the mitochondrial membrane; a function that is opposed by Bcl-2 activation (Oltvai et al., 1993). The expression of Bax is normally held controlled by Bcl-2 but the other member of Bcl family the BIM antagonize the interaction and allow Bax to initiate apoptosis. The members of the BH3only sub-family include BID and BAD proteins (Bcl-2 antagonist of cell death) activities usually kept in check by controlling their expression during transcription or by post-translational modification to maintain the normal cellular functions (Yip and Reed, 2008).

13.12 Regulatory mechanism of apoptosis in animal cells In the early development of organisms, the conception of haploid spermatozoon and ovum to form zygote is primarily intricate with the signaling cascade for survival and also for the death program of the cells to clearly frame the structure of an organism during morphogenesis. However, the predictability of the death for better cause initiates from existing examples of embryonic chick wings development and the death of intersegmental muscles in moths and similar organisms. Regulation of apoptosis in animal models is explained from fertilization to the maturation to clarify the interrelation of cells during animal development. A study using zebrafish eggs exposed with apoptotic-causing drug cycloheximide actually shows the necrotic death instead of the apoptotic death of an older embryo, but the same cells show activated caspase three that is in a program of necrosis (Negron and Lockshin, 2004). Later clarifies that although the cells function in their respective pathway, the execution of such signals by surrounding cells is delayed and accumulation leads to inflammatory or other responses, and in the case of egg embryo the surrounding phagocytes are limited and so more apoptotic cells are accumulated and they even degraded (Ledda-Columbano et al., 1991; Negron and Lockshin, 2004). The immortalized cell lines are used as a model system to understand the various complex biological and cellular events. In the case of tissue culture cell lines, the fate of a cell is always undergoing necrotic cell death, because of simple mechanism that, if a cell is not phagocytosed by macrophages after degradations by any mediated pathway the cell will ultimately be lyzed. Hence, compared to cell line studies, the tissue or animal models are best suitable in understating the programmed-cell-death as they represent physiological conditions (Lockshin and Zakeri, 2004; Yuan et al., 2003).

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As part of morphogenesis, aging in several tissues shows loss of cells that is contributing to programmed-cell-death or similar kind of processes. In mammals, sarcopenia, an aging-associated skeletal muscle weakness shows symptoms of reductions in muscle fiber size and loss of fibers (Marzetti et al., 2008). However, a study in rodents suggests the mitochondrial malfunction and abnormal apoptosis signaling are responsible for sarcopenia (Marzetti et al., 2013) as the expression of apoptotic markers correlates with aging and sarcopenia condition in muscle tissues. Similarly, the involvement of apoptosis in the aging of rat gastrocnemius muscle tissue showed increased levels of proapoptotic Bax, decreased antiapoptotic Bcl-2, and subsequent activation of caspase-3 leading to DNA fragmentation (Song et al., 2006). In another rodent model altered Bax/Bcl-2 ratio, the release of cytochrome-c and caspase3 activation showed a loss of myocytes in the aging heart (Kwak, 2013). In a study during simulation of microgravity, the effectors of the apoptotic pathway like caspase-8 and -3 on the transcription factor NF-kB signaling molecule in mouse testis characterized to correlate the development of reproductive organ and its function and showed simulated microgravity activated apoptosis and NF-kB levels (Sharma et al., 2008). The normal or uncontrolled cellular proliferation can be inhibited by apoptosis and is induced by toxic metals (Kiran Kumar et al., 2016). Such metal toxicity on the cellular function, including apoptosis, is characterized using uranium induces apoptotic lung epithelial cells with an increase in reactive oxygen species as a consequence of oxidative stress (Periyakaruppan et al., 2009). Caspases (cysteinyl, aspartate-specific proteases), a family of cysteine proteases, are the central regulators of the apoptosis pathway. The initiator caspases produced as inactive proteins (zymogens or procaspases) and activate themselves through auto-proteolysis that is facilitated by the interaction with adapter molecules (Nicholson, 1999). Once the initiator caspases get activated, they cleave off the executors caspases to break specific cellular substrates resulting in apoptotic cell death (Stennicke and Salvesen, 2000). During initiation, the caspases 2, 8, 9, 10, 11, and 12 are closely associated with pro-apoptotic signals. Once activated by stimulation and induction, they activate downstream effector caspases 3, 6, and 7, which later execute apoptosis by cleaving cellular contents. This caspases activity is a hallmark in the apoptosis process that is responsible for chromatin condensation, plasma membrane asymmetry, and cellular blebbing (Pistritto et al., 2016). Activation of the receptor-mediated death domain (FADD/TRADD) leads to the activation of caspase-8 and -10 from their inactive state. On another side, cytochrome-c released from ruptured mitochondria is associated with APAF-1 and procaspase nine, forming an apoptosome complex to activate the caspase-9 and further activation of caspase three leading to genome fragmentation. As an early marker of an apoptotic cell, the cleavage of genomic DNA into smaller fragments are observed in several numbers of cells/tissues types, including, breast cells, thymocytes, leukemia cells, and human T lymphoblastoid cells. Following DNA damage or by a variety of cellular stress, the tumor suppressor p53-transcription factor molecule is capable of inducing apoptosis with activation of intrinsic pathway and transcriptional activation of many apoptosis-inducing genes and p53-TF is also shown to involved in the DNA repair mechanism (Agarwal et al., 1998; Geske et al., 2001; Hegde et al., 2016).

13.13 Apoptosis deregulation and diseases The deregulation of apoptosis leads to several cellular complications, including harmful symptoms, with untreatable disease outcome in humans, as well as other animals. Regulation of apoptosis is a complex and fine-tuning process to maintain normal physiologic cell numbers, and failure in such programs may increase the cell population, and as a consequence leads to tumor development. The tumor progression is often associated with the elimination of apoptosis, which further causes hyperplasia and malignancy (Hengartner, 2001). Tumor progression is associated with the uncontrolled-cell-proliferation and can be avoided by inducing apoptosis through a number of mechanisms. In the case of neurodegenerative diseases such as Parkinson’s or Alzheimer’s disease, the apoptosis is because of much cell death and the progressive loss of neurons. Neurodegenerative diseases or AIDS diseases are associated with too much of apoptosis and in cancer with too little apoptosis due to mutations of tumor suppressors or overexpression of antiapoptotic proteins. The premature death of adult neurons with functional deficiency leads to neurodegeneration, and the remaining neurons lose the capacity for regenerating neurons in Alzheimer’s, Huntington’s, and Parkinson’s disease conditions (Raoul et al., 2002). Whereas in autoimmune disease conditions, deficiency in serum proteins or receptors, which mediate the phagocytic clearance of apoptotic cells, increases the risk of autoimmunity with the altered inflammatory response (Jin and El-Deiry, 2005; Voll et al., 1997). Studies also suggest that a disruption of normal autophagy or mitochondrial-dependent mitophagy contributes to mitochondrial dysfunction and thereby promotes programmed cell death (Ghavami et al., 2014; Hroudov et al., 2014; Tower, 2015). The inhibition of apoptosis or its resistance toward apoptosis plays a major role in tumor promotion. The mechanism through which the evasion of apoptosis occurs is due to a disrupted balance between pro- and antiapoptotic proteins or through reduced caspase function and by impaired death receptor signaling. Evading cell death is one of the major changes that lead to metastasis and malignant transformation

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(Babu et al., 2013; Hanahan and Weinberg, 2000). Further, there are numerous ways by which a malignant cell can resist apoptosis, and cancers are caused due to a series of genetic alterations during which the normal cell is transformed into a malignant cell. The common observation during apoptosis is the reduced expression or mutation of tumor suppressor p53 gene results in tumor growth (Bauer and Helfand, 2006) and inactivation of p53 has been linked with the development of many cancers irrespective of the mechanism (Morton et al., 2010; Nagesh et al., 2017). The p53 can trigger apoptosis, as well as resist apoptosis by participating in a DNA repair mechanism to specifically target tumor cells (Baritaki et al., 2011; Jensen et al., 2008). Cell cycle plays an important role in the growth and development of all the organisms, but often apoptosis is a consequence of irreversible cell cycle arrest due to altered regulation of cell cycle regulators (Naveen Kumar et al., 2019; Nurse, 2000). Drugs that recover the normal apoptotic pathways in disease state tissue or in cells need to be more potential in treatment, which are specifically dependent on aberrations in the apoptotic pathway. Receptor and protein/gene oriented apoptosis inducer is currently being explored for cancer drug discovery for an alternative therapy (Fesik, 2005). Most anticancer drugs, at present, in clinical studies are trying to exploit the normal apoptotic pathways to trigger the death of cancer cells. However, defects in the cell death cascade may be resulting in drug resistance and so limiting the efficacy of therapies so far. Hence, a better understanding of the apoptotic cell death signaling pathways may improve the efficacy of cancer therapy and bypass resistance (Pistritto et al., 2016). Hence, it is of great importance to understand the mechanism of apoptosis in diseased cells, which facilitates a better understanding of the pathogenesis of a particular disease and helps in framing active therapeutic modalities to cure such abnormalities.

13.14 Methods of apoptosis detection Apoptosis process has a multiple biological significance and actively participates in differentiation, proliferation, regulating the function of the immune system, in the elimination of defected harmful cells and to carry out the normal function in cell systems (Babu et al., 2013; Naveen Kumar et al., 2019; Patil et al., 2016). Knowing its actual role in the game of cell survival or death requires more attention and methods to analyze its play according to the situation. Over time various methods of detecting apoptotic cells are explored and made available to this generation. Hence, applying the right techniques to analyze and detect apoptotic cells along with its characters are required to understand its behavior in biological events. Usually, relevant morphological characteristics and detectable markers of apoptotic pathways are selected during molecular assays/analysis. The detection methods broadly focus and depend on cell proliferation, cytotoxicity, membrane integrity, DNA fragmentation, mitochondrial redox status, immunological reactions, and mechanism/cascade-based assays (Banfalvi, 2017; Martinez et al., 2010). Microscopic observation identifies the early process of apoptosis, showing cell shrinkage and membrane damage routinely by using nuclear-stained light microscopy. The light microscopy method is fairly a reliable and inexpensive method for the detection of apoptotic cells but lacks quantitative measurement and reproducibility (Ghobrial et al., 2005). However, the electron Microscopy defines the subcellular changes better and shows the most noticeable changes like chromatin condensation, nuclear material aggregation, plasma membrane blebbing, and separation of cell fragments into apoptotic bodies during budding. This procedure is time taking and more expensive and it requires laborious preparation with instrument maintenance (Archana et al., 2013; Yasuhara et al., 2003). In gel electrophoresis, the characteristic of apoptotic DNA fragments following sequential degradation are produced in the form of fluorescent bands under UV exposure. It is easy, sensitive, quantitative, and precise in the determination of cell death and DNA damage as DNA fragments are considered as a more reliable biochemical marker for apoptosis. Further conventional and advance methods of gel electrophoresis are also used in specific molecular analysis (Gavrieli et al., 1992; Yasuhara et al., 2003). Flow cytometry is used for the accurate quantification and to distinguish apoptotic from nonapoptotic cells by using fluorescent DNA staining dyes (Kim et al., 2007). This technique allows counting and sorting of cells and their specific cell cycle phase with simultaneous multiparametric analysis of both viable and fixed single cells while flowing through an optical detector. Intact tissues require pretreatment and are time consuming during multiple step quantification procedure (Martinez et al., 2010). In situ labeling makes use of radioactive or nonradioactive labeling of the free ends of the DNA fragments analyzed by TUNEL assay is more sensitive. It enables in situ visualization of the process at the single-cell level. The reactions are based on the direct labeling of 30 -hydroxyl termini of DNA breaks, and the cuts measured are identifiable at the molecular level (Banfalvi, 2017). Immunodetection, immunocytochemistry, and immunohistochemistry methods of detection of apoptotic cells are performed using antibodies against an exclusive range of protein or substrates most important one are caspases, receptors, membrane, and apoptotic markers. These methods are highly sensitive and analyze specific quantitative status of proteins in cells (Ray et al., 2000).

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13.15 Conclusion Cell signaling and apoptosis are very important aspects of the operation of the cellular system, and it is important to compile the exiting information, and making available for the scientific community is much essential. Cell signaling in animals is a complex process, taking through various processes like autocrine, paracrine, endocrine, etc., which are coordinated through signaling receptors such as intracellular receptors and cell surface signaling receptors (GPCRs, ligandgated ion channels, enzyme-linked receptors, RTKs). All these cellular events regulate the cells and bring the physiological response, any deviation or defects in the above process leads to diseases. In animal cell signaling, “second messengers” play a pivotal role in amplifying the signal strength to provide changes in cell activity through activating various cellular pathways. Many pathways have been elucidated in animals such as WNT pathway, NF-kB pathway, nitric oxide pathway, and nuclear receptor pathway, and these pathways are activated, based on specific stimulus, thereby controlling the cell growth, physiology, and needs of the cell. Since these pathways have complex networks, many biological pathway database networks will provides us the information regarding the pathway network and signaling between the molecules. Through computational modeling approach, many studies are underway to provide the systematic networking pathways of various cellular pathways. Cell signaling brings various responses, such as change in gene expression, an increase in cellular metabolism, cell growth, and cell death. Cell signaling or signal transduction varies from stage to stage in the cell, and therefore a thorough understanding of the signal transduction process aids in the disease diagnosis and developing of treatment modalities. Apoptosis is a natural cell death in multicellular organisms and referred as programmed cell death. Cell death plays a vital role in the development of organism and the stringent process of cell death occurs through different mechanisms such as apoptosis, autophagy, and necrosis. Apoptosis process has a multiple biological significance and actively participates in differentiation, proliferation, regulating the function of immune system, in the elimination of cells during normal embryonic development, and in the elimination of defected harmful cells in different cell systems. Apoptotic cells and their properties found to be similar in all animal cell/tissue types and results in the loss of membrane integrity, leakage of cellular components, and consecutive nuclear degradation eventually form a apoptotic bodies and captured inside the macrophages by the process of phagocytosis that is induced by physiological or pathological environmental stimuli by avoiding cross-inflammatory responses. Mainly apoptosis is mediated by extrinsic or intrinsic pathways and caspases, pro- and antiapoptotic regulators are the key agents in determining the cell to undergo apoptosis. Regulation of apoptosis in animal models is explained from fertilization to the maturation to clarify the interrelation of cells during animal development. Further, compared to cell line studies the tissue or animal models are best suitable in understating the programmed cell death as they represent physiological conditions. Progression of apoptosis is a highly coordinated process and failure in the program leads to tumor development, which is associated with the uncontrolled cell proliferation. The detection methods of apoptosis broadly focus and depend on the cell proliferation, cytotoxicity, membrane integrity, DNA fragmentation, mitochondrial status, immunological reactions, and mechanism/cascade-based interactions. Hence, it is of great importance to understand the mechanism of apoptosis in diseased cells, which facilitates a better understanding of the pathogenesis of a particular disease and helps in framing active therapeutic modalities to cure such abnormalities.

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Chapter 14

Molecular Network for Management of Neurodegenerative Diseases and their Translational Importance using Animal Biotechnology as a Tool in Preclinical Studies Nibedita Naha Biochemistry Division, ICMReNational Institute of Occupational Health (NIOH), Ahmedabad 380016, Gujarat, India

Abbreviations ND Neurodegenerative diseases/disorders AD Alzheimer’s disease PD Parkinson’s disease HD Hungtinton’s disease ALS Amylotrophic lateral sclerosis IT15 Interesting transcript 15/Huntingtin gene APP Amyloid precursor protein MTHFR 5,10-methylenetetrahydrofolate reductase PINK1 PTEN-induced putative kinase 1 DJ-1 Protein deglycase PARK7 PD protein 7 LRRK2 Leucine-rich repeat kinase 2 ATP13A2 ATPase cation transporting 13A2 ROS Reactive oxygen species RNS Reactive nitrogen species SIRT Sirtuin ER Endoplasmin reticulum nNOS Neuronal nitric oxide synthase PARP Poly(ADP-ribose) polymerase GAPDH Glyceraldehyde 3-phosphate dehydrogenase CHIP C-terminus of heat shock cognate (Hsc 70)-interacting protein HSP Heat shock protein 4E-BP Eukaryotic initiation factor 4E (eIF4E)-binding protein TRAP1 Tumor necrosis factor receptor associated protein 1 HtrA2 High temperature regulated serine protease A2 NCK Sodium/calcium/potassium exchangers CNS Central nervous system FBP-1 Far upstream sequence element-binding protein 1 AIMP2 Aminoacyl-tRNA synthetase complex interacting multifunctional protein 2 DAXX Death-associated protein 6 ASK1 Apoptosis signal-regulating kinase 1

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PPD Parkin-PINK1-DJ-1 complex PARK9 PD protein 9 gene GBA b-Glucocerebrosidase SERCA Smooth ER-calcium pump MPTP 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine MPP+ 1-methyl-4-phenyl-pyridium AIF Apoptosis-inducing factor Htt Huntingtin protein IL Interleukin TNF Tumor necrosis factor TGF Transforming growth factor CD206 Mannose receptor NF-kb Nuclear factor kappa B NMDA N-methyl-D-aspertate TLR Toll like receptor AMPA a-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid Hdh HD gene homolog FTD Frontotemporal dementia SOD1 Superoxide dismutase 1 C90RF72 Chromosome 9 open reading frame 72 TARDPB Transactive response DNA binding protein 43 FUS Fused in sarcoma gene VAPB Vehicle-associated membrane protein B VCP Valosin-containing protein UBQLN2 Ubiquilin 2 ATXN2 Ataxin-2 gene NEFH Heavy chain neurofilament GRN Progranulin CHCHD10 Coil-helix-coiled-coil-helix domain CSF Cerebrospinal fluid HSF1 Heat shock factor 1 IB Inclusion body (IB) MitoQ Mitoquinone mesylate PGC1a Peroxisome proliferator-activated receptor gamma coactivator 1a Nrf2 Nuclear factor erythroid 2-related factor 2 AMPK AMP-activated protein kinase NMN Nicotinamide mononucleotide BBB Blood-brain-barrier mTOR Mammalian target of rapamycin ETC Electron transport chain DRP1 Dynamin-related protein-1 MSC Mesenchymal stem cells ASC Adipose tissue-derived MSC.

14.1 Introduction Neurological disorders represent a significant health problem worldwide, which costs an economic burden in the society. For example, the economic burden of autism in France costs nearly 1.4 billion Euros per year. Higher life expectancy further increases the prevalence of age-related neurodegenerative diseases/disorders (ND) such as, Alzheimer’s disease (AD), Parkinson’s disease (PD), Huntington’s disease (HD), amyotrophic lateral sclerosis (ALS) and so on. Around 7e10 million people worldwide have PD, and 4% of them are diagnosed at the age of below 50 years (http://parkinsonsnewstoday.com/ parkinsons-disease-statistics). Approximately 50 million people suffer from AD and nearly 10 million new cases are enrolled each year (http://www.who.int > news-room > fact-sheets > detail > dementia). HD affects one in every 10,000 or around 30,000 people in the USA; also 150,000 or more people are at a risk of developing the disease (http://www. medicalnewstoday.com > articles) although the prevalence is remarkably lower in Asian populations compared with Western Europe, North America, and Australia, might be partly explained by the average CAG repeat lengths and frequency of different Huntington gene haplotypes in the general population (Baig et al., 2016). Although ALS is relatively rare, but its

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socioeconomic impact is huge because ALS cases across the globe will increase from 222,801 in 2015 to 376,674 in 2040, representing an increase of 69% due to the aging of the population, particularly among developing nations (Arthur et al., 2016). Overall, ND is a threat because it decreases the quality of life and morbidity last throughout the lifetime of an individual, even if first observed during pregnancy or early childhood. Commonly, all these above-mentioned disorders manifest dysfunction of communication of the central nervous system (CNS), especially connectivity among different brain parts such as, hippocampus, midbrain, cerebral cortex, thalamus, etc. (Piazza-Gardner et al., 2013; Susick et al., 2016). However, the increasing prevalence of ND requires novel approaches through molecular mapping using genetically engineered preclinical animal models of translational inputs. Further, AD, PD, and ALS are sporadic: only 4%e5% cases are caused by genetic mutations (Chen et al., 2012; Lee et al., 2012; Kiernan et al., 2011), whereas HD is caused by mutation of one particular gene, “interesting transcript 15” (IT15) (Gusella et al., 1993; Andrew et al., 1993). Therefore, a powerful tool for understanding such ND is to develop a model that represents the hallmark of the disease characteristics. Thus, identification of genetic mutations for different ND is made by several transgenic mice models through expression of mutant proteins, resulting in development of various suitable small animal models in reality, either from expression of mutant genes under different promoters, or by using other transgenic approaches. In contrast, sometimes mouse models fail to represent the full range of neuropathology seen in the patients’ brain such as, absence of overt neurodegeneration in rodents that express transgenic mutant proteins under the control of different promoters, might be due to shorter life spans and differential aging processes of smaller animals. The brain development period is also crucial in this regard, which takes more than 150 days in humans in contrast to 21 days in rodents (Li and Li, 2015). Although studies indicate 1 rat day is equivalent to 34.8 human days, or 1 rat month is comparable to 3 human years (Sengupta, 2013); and 1 mice day is approximately 40 human days, or 1 human year corresponds to 9 mice days (Dutta and Sengupta, 2016); but rapid brain development in rodents may render neuronal cells resistant to misfolded protein-mediated neurodegeneration. Besides rodent models, fruit fly, nematode worm, baker’s yeast are also used for studying ND in humans. But they can be challenged due to drastic differences in physiology and underlying mechanism between species. Moreover, most ND involves an aging component that takes years to grow in humans, and hence, these animal models may not always be appropriate. Currently, large animal models are used such as, pigs, sheep, and non-human primates for HD, ALS, and PD because of mimicking human scenario as seen in the patients. Recent developments in genome editing with new technologies also make it possible to establish large animal models to investigate ND (Niu et al., 2014; Wan et al., 2015). However, large animals need to be analyzed critically prior to birth of offspring, which arise ethical issues and might confound the phenotype of the parent animal. Hence, genetic engineering of large animals for translational research in ND is a concern worldwide. Overall, till date pathogenesis of most of the ND is not clearly known, and therefore, effective treatments are not available although some genetically engineered targets open new therapeutic strategies; and preclinical animal models might provide insights into the pathophysiology of the disease mechanism along with the efficacy of the potentiality of new drug therapies prior to clinical trial in human. With this aim, the current book chapter briefly highlights the recent advancement of knowledge regarding the pathogenesis of four ND, such as, AD, PD, HD, and ALS, and molecular mapping of drug targets (proteins, genes, etc.) along with translational importance of preclinical animal models. For this purpose, data were searched through Pubmed, Medline, Toxline, Research Gate, and Google using numerous combinations of terms pertaining to different aspects of genetic engineering in the pathogenesis of ND and translational values in animal biotechnology.

14.2 Pathogenesis and Molecular Mapping of Neurodegenerative Diseases 14.2.1 Alzheimer’s Disease (AD) AD is characterized by loss of memory (dementia), the inability to learn new things and calculation, loss of language function, as well as depression, and delusions, which affect the social life of the patients. Within the first 5e10 years, AD becomes progressive and fatal due to complications of chronic illness. Based on clinical data, 13% of AD patients are over 65 years, and 45% are over 85 years. AD generally occurs in two ways: extracellular deposition of amyloid-b (i.e., part of senile plaques, consider primary) and intracellular accumulation of tau protein (i.e., part of neurofibrillary tangles, consider secondary). Both tau and amyloid-b are insoluble. Amyloid-b toxicity in neurons includes loss of long-term potentiation, synaptic damage, and cell death, selectively in temporal lobe, hippocampus, and cortex, mediated by free radicals when the soluble form of amyloid-b form complexes with zinc, copper, and iron (Aso and Ferrer, 2013). Senile plaques, also called Alzheimer’s plaques, are spherical, 100 m in diameter and of two types: diffuse and neuritic plaques containing degenerating neural processes with tau filaments, reactive astrocytes, and microglia. In transgenic AD models, severe

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neurological deficits are observed in the absence of amyloid deposition in tissue (Kitazawa et al., 2012). Based on this hypothesis, transgenic mice that are overexpressing a mutant amyloid precursor protein (APP) and developing AD neuropathology, exhibit a great translational value. Active immunization of young animals with amyloid-b and passive immunization with its antibody prevent the development of AD, whereas in older animals, it reduces the extent and severity of the disease pathogenesis (Gallardo and Holtzman, 2017). However, based on these findings, phase II trials of a human vaccine consisting of synthetic amyloid-b are unable to clear due to autoimmune meningoencephalitis in some patients (Cribbs, 2010; Wisniewski and Konietzko, 2008). In AD, further, over-phosphorylated microtubule-associated protein tau is aggregated as an insoluble twisted filamentous polymer (i.e., neurofibrillary tangles) in the neuronal body specifically in hippocampus and cerebral cortex, leading to the displacement of cell organelles and impairment of cell functions. Also, by distorting microtubule spacing, it interferes with the axonal transport of nutrition to neuronal branches and dendrites. The overall event is manifested by neurofibrillary degeneration although tau mutation is not found in AD (Braak and Braak, 1991). Transgenic mice study suggests the passage of abnormal tau through synapses spread the disease pathology to anatomically linked areas of the brain (Mudher et al., 2017; Albert et al., 2019). In advanced AD, hippocampus contains extracellular neurofibrillary tangles with tau embedded in neurophils like, fossilized skeletons of neurons or ghost tangles but the mechanism of tau aggregation is unclear although the development of neurofibrillary tangles is independent of amyloid-b, and the cognitive decline correlates much with the neurofibrillary tangles load than that of the number of amyloid-b plaques (Nelson et al., 2012). In contrast, genetic and environmental risks for AD are strongly associated with amyloid-b (Zawia and Basha, 2005). In very severe cases, both amyloid-b plaques and tau are found in brain stem (Uematsu et al., 2018). The role of contributing factors to aggravate AD pathogenesis is also identified such as, chronic damage by free radicals, excitotoxicity, non-enzymatic glycation of proteins, etc. contribute to neural/synaptic loss; neuroinflammation due to high level of serum acute-phase protein APP, deposition of senile plaques; microglial accumulation, which is the source of cytokines and free radicals, and presence of complement components; oxidative mutation of mitochondrial DNA, etc. (Mattson, 2003; Prentice et al., 2015). These factors are accelerated with age and senile plaques, which is a mitochondrial poison and free radical generator. Further, insulin receptor density is highest in olfactory bulb, hypothalamus, hippocampus, cerebral cortex, striatum, and cerebellum, suggesting insulin signaling through insulin-insulin receptor-AKT and MAPK pathways play a diverse role in the development and maintenance of synapses, dendritic spine formation, and neural survival in the brain. Type 2 diabetes, obesity, low insulin, and insulin resistance in the brain, impairment of energy metabolism, and insulin receptor signaling pathways in neurons are also considered as risk factors for AD (Arnold et al., 2018). Insulin resistance contributes to AD via a number of mechanisms, including the promotion of disease-specific pathological lesions and high neuronal vulnerability (Arnold et al., 2008). In contrast, the administration of intranasal insulin also improves cognitive function in the patients with AD and amyloid-b features in mice model of AD (Arnold et al., 2008). Moreover, traumatic brain injury; glutamate excitotoxicity; apoptosis and low levels of ATP; ischemia (i.e., cerebral atherosclerosis); loss of cholinergic neurons and hence, reduction in acetylcholine; increase level of homocysteine, a risk factor for stroke along with polymorphisms of 5,10-methylenetetrahydrofolate reductase (MTHFR), an important enzyme in folate metabolism, as well as decrease level of dietary folate and high cytosolic calcium potentiate the pathogenesis of AD (Moretti and Caruso, 2019; Prentice et al., 2015; Mattson, 2003). However, AD pathogenesis in nondemented older people (Harman, 2006) still creates a dilemma of the neuropathologists.

14.2.2 Parkinson’s Disease (PD) PD is the most common movement disorder by the death of dopaminergic neurons, manifested by rest tremor, rigidity, bradykinesia, and postural instability. Although it is believed that PD emerged as a result of industrial revolution, evidence showed the existence of a disease called “kampavata,” consisting of shaking (kampa) and lack of muscular movement (vata) in ancient Indian medical system Ayurveda, as long as 4500 years ago (Manyam, 1990), which was treated by Mucuna pruriens plant that was later discovered to contain levodopa, today’s main treatment regimen of PD (Katzenschlager et al., 2004). While the exact cause of PD is still unknown, but advancement in possible underlying mechanisms (Jankovic and Sherer, 2014) clears the anatomy and function of basal ganglia, substantia nigra pars compacta, midbrain, and thalamus in PD pathogenesis. Dopamine depletion from basal ganglia leads to major disruptions of the neural circuit in thalamus, midbrain, and cerebral cortex (motor cortex), manifested by Parkinsonian signs such as, bradykinesia. About 5%e20% of PD patients have a familial form of the disease with an autosomal-dominant pattern of inheritance (Mullin and Schapir, 2015). Additionally, the incidence of PD is greater in the family members of different generations than age-matched controls (Olanow and Tatton, 1999). Genetic analysis reveals PD-associated genes like, a-synuclein,

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parkin, PTEN-induced putative kinase 1 (PINK1), protein deglycase (DJ-1; also called PD protein 7 or PARK7), leucinerich repeat kinase 2 (LRRK2) and ATPase cation transporting 13A2 (ATP13A2) play significant role in common and divergent mechanisms of PD pathogenesis (Gupta et al., 2008; Schapira, 2008). Although a-synuclein is associated with membranes, synaptic vesicle recycling, dopamine neurotransmission, and lipid interactions (Burré, 2015), its physiological function is not fully known. a-Synuclein is a major structural component of Lewy bodies, the pathological hallmark of PD (Xu and Pu, 2016). Identification of a-synuclein gene abnormalities indicates the existence of abnormal protein encoded by this gene in PD pathophysiology. Not only unfolded (native state) or mutated a-synuclein monomers (A53T, E46K, A30P) form toxic intermediates such as, oligomers and fibrils (Lashuel et al., 2013), but also triplication of this gene lead to PD; might be due to high steady-state level of wild-type a-synuclein (Bridi and Hirth, 2018). Reactive oxygen species (ROS), reactive nitrogen species (RNS), aging, etc. play a crucial role in a-synuclein aggregation during PD. ROS/RNS production, disruption of macroautophagy, mitochondrial dysfunction, and proteasome inhibition can also trigger by mutant or aggregated a-synuclein in dopamine-related ND like, PD (PozoDevoto and Falzone, 2017; Protter et al., 2012). Apart from a-synuclein, histone deacetylase and sirtuin (SIRT) 2 may contribute to PD pathogenesis. ER stress, neuronal nitric oxide synthase (nNOS) activation, DNA damage, poly (ADP-ribose) polymerase (PARP) activation, and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) modification, etc., are also linked with PD Genes (Akbar et al., 2016). Further, LRRK2 gene is localized in cytoplasm and associated with membranous structures, including mitochondria, ER, and synaptic vesicles. Mutation of LRRK2 results in kinase-dependent neuronal toxicity regulates by C-terminus of heat shock cognate (Hsc 70)-interacting protein (CHIP) and heat shock protein (HSP) 90 (HSP90) chaperones through ubiquitin-proteasome system (Chen and Wu, 2018), and in a-synuclein pathology but controversy still exists (Henderson et al., 2018). Functional studies implicate the role of LRRK2 in neurite outgrowth and endocytosis of synaptic vesicles. There are many putative substrates of LRRK2 present in CNS, including moesin, eukaryotic initiation factor 4E (eIF4E)binding protein (4E-BP), which suggest the involvement of LRRK2 in the translation of wrong protein, mitochondrial dysfunction, etc. during the development of PD pathogenesis (Biskup and West, 2009). PINK1 is another gene, might be a form of a mitochondrial kinase that acts upstream of parkin in PD pathogenesis cascade (Abeliovich, 2007). Mitochondrial chaperone tumor necrosis factor receptor associated protein 1 (TRAP1) and high temperature regulated serine protease A2 (HtrA2) are putative PINK1 substrates that regulate mitochondrial-dependent cell death signaling and calcium efflux via sodium/calcium/potassium exchangers (NCK), which might explain the functional loss of specific CNS neurons in PD due to mutation of PINK1 in different preclinical genetic engineered models (Celardo et al., 2014). Mutation of parkin gene and posttranslational modifications of the protein by ROS/RNS blocking the functional activity of parkin as E3-ubiquitin ligase (Meng et al., 2011; Chakraborty et al., 2017), leading to accumulation of far upstream sequence element-binding protein 1 (FBP-1), aminoacyl-tRNA synthetase complex interacting multifunctional protein 2 (AIMP2), and others that involve in mitochondrial dysfunction and CNS toxicity in ND (Tan et al., 2019; Brahmachari et al., 2017), impairment of intracellular trafficking of proteins and formation of inclusion body (IB) (Hunn et al., 2015). In the genetic model, parkin acts downstream of PINK1 and plays a critical role in the clearance of mitochondria (Mouton-Liger et al., 2017; Celardo et al., 2014). DJ-1, a molecular chaperone, shows multiple functions such as, regulates ROS levels by acting as an atypical peroxiredoxin-like peroxidase, modulates RNA metabolism and gene transcription, and might be neuroprotective in PD (Ariga et al., 2013). DJ-1 mutation causes autosomal recessive early onset of PD in both the sporadic and familial forms; DJ-1 inclusions present in a-synucleopathies and tauopathies (http://www.ncbi.nlm.nih.gov/gene/11315). DJ-1 binds to histone chaperone death-associated protein 6 (DAXX) and apoptosis signal-regulating kinase1 (ASK1), resulting in inhibition of ASK1 activity leading to cell death (Junn et al., 2005; Karunakaran et al., 2007). It has been believed that DJ-1 involves in parkin-PINK1-DJ-1 complex (PPD) and promotes the degradation of heat shock misfolded proteins (Xiong et al., 2009). ATP13A2 is a large lysosomal P-type ATPase encode by PD protein 9 gene (PARK9), which along with PD susceptibility gene encoding b-glucocerebrosidase (GBA), involves in the solubilization of a-synuclein aggregates (Siebert et al., 2014; Schultheis et al., 2013). A new PD mouse model with loss of endolysosomal protein ATP13A2 causes behavioral, neuropathological, and biochemical changes similar to those present in human subjects with ATP13A2 mutations and raises questions about the consequences of endolysosomal dysfunction in PD (Kett and Dauer, 2016). Although the functional studies of DJ-1 and ATP132A2 are at a primitive stage, but based on the above information, both might be future promising gene targets in the treatment of PD pathogenesis. Diverse environmental factors along with genetic risk factors increase PD pathogenesis although a detailed mechanism of action is yet to be investigated. Overall, mitochondria are the key target in PD pathogenesis, and mitochondrial dysfunction causes the death of dopaminergic neurons. Because dopaminergic neurons use L-type calcium channels (Cav1.3) for pacemaking, resulting in high ATP consumption and calcium influx to ER via high-affinity smooth ER-calcium pump (SERCA); calcium flows

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back to the cytoplasm through inositol trisphosphate and ryanodine receptors, leading to the formation of the calciumrich zone near mitochondria, which uptake calcium via calcium uniporter unlike to sodium-calcium exchanger that mediates calcium efflux from mitochondria (Zaichick et al., 2017). Due to unique Cav1.3 channels, dopaminergic neurons are vulnerable to environmental toxins such as, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) (Surmeier et al., 2011; Choi et al., 2015). MPTP selectively kills dopaminergic neurons by blocking mitochondrial complex I activity through the accumulation of 1-methyl-4-phenyl-pyridinium (MPPþ) in mitochondria by dopamine transporter when MPTP is metabolized by monoamine oxidase B (Surmeier et al., 2011; Choi et al., 2015). This leads to depolarization of the mitochondrial membrane and opening of permeability transition pore, resulting in the release of cytochrome C and apoptosis-inducing factor (AIF) in cytosol, causing untimed death of the dopaminergic neurons through caspase-dependent/independent pathway (Gupta et al., 2008; Schapira, 2008). Microglial activation also contributes to dopaminergic neuron death (Ferreira and Romero-Ramos, 2018). Based on this theory, most of the preclinical experimental models of PD are developed.

14.2.3 Huntington’s Disease (HD) HD is an incurable, adult-onset, slow progressive autosomal dominant inherited disorder associated with cell loss within a specific subset of neurons in striatum, cerebral cortex, and other brain parts, caused by an expanded CAG triplet repeat in IT15 gene located on chromosome 4p16.3, which encodes an abnormal polyglutamine expansion close to the aminoterminal of huntingtin (Htt) protein (McDonald, 1993; Jimenez-Sanchez et al., 2017). The overall situation is manifested by involuntary movements (motor abnormalities), dementia and cognitive-behavioral changes (Bates et al., 2015). Huntingtin plays a role in protein trafficking, vesicle transport, postsynaptic signaling, transcriptional regulation, and apoptosis. Thus, a loss-of-function of normal Htt protein and a toxic gain-of-function of mutant Htt aggregates (the hallmark of HD) contribute to the disruption of multiple intracellular pathways, involving excitotoxicity, dopamine deregulation, metabolic impairment, mitochondrial dysfunction, oxidative stress, apoptosis, and autophagy during the progression of the disease (Jimenez-Sanchez et al., 2017) (Table 14.1). Intranuclear inclusions and protein aggregates in striatal and cortical neurons are also observed in the HD patients. According to polar zipper model, expanded polyglutamine tail destabilizes the normal tertiary Htt protein conformation and produces abnormal protein-protein interactions with other polyglutamine-bearing molecules of the mutant and wild-type Htt, resulting in insoluble b-pleated sheets that form polar zipper structures via hydrogen bonding (Perutz et al., 1994; Stott et al., 1995). Transglutaminase model states the involvement of transglutaminases in cross-linking of glutamine residues during the formation of protein aggregates in HD, as Htt is a transglutaminase substrate; thus transglutaminase-mediated cross-linking increases with the length of the polyglutamine stretch (Kahlem et al., 1998). Transglutaminase activity also increases in brains of the HD patients (Karpuj et al., 1999). Posttranslational modification of different serine residues (i.e., serine 421, 434, 513, and 536), reversible binding of small ubiquitin-related protein modifier, palmitoylation, etc. of Htt plays a crucial role in intracellular trafficking of the molecule in patient’s brain as a part of toxic gain-of-function (Rangone et al., 2004; Luo et al., 2005; Schilling et al., 2006; Yanai et al., 2006). Cell-cell interaction is also important in the normal function of neural circuits among different brain regions. Glial cells that constitute 90% of cells in brain, and provide nutrition, growth factors, and structural support to neurons, are

TABLE 14.1 Gradation of HD and progressive degeneration of different brain parts. HD grade

Affected brain parts

Characteristic features

Grade 1

Caudate nucleus

Atrophy of tail; Neuronal loss and astrogliosis in the head (50% loss), tail, and to some extent in the body

Grade 2

Striatum

Gross striatal atrophy more pronounced than grade 1 but no non-striatal atrophy

Grade 3

Striatum; globus pallidus; cerebral cortex (layer III, V, VI); subthalamic nuclei; hypothalamus; thalamus; cerebellum

Severe gross striatal atrophy

Grade 4

Atrophy of striatum (95%), medium spiny (motor) neuron and white matter (of brain parts); Loss of GABAergic/dopaminergic inputs; Loss of brain weight

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neuroprotective by removing excess excitatory neurotransmitters from the extracellular space. This is, in particular, relevant to the neuropathology of HD since excitotoxicity is a long-standing theory to account for the disease pathogenesis (Li and Li, 2006). Mutant Htt is also expressed in glial cells of both HD mice and HD patients, which reduces glial glutamate level and confers protection against Htt-mediated neurotoxicity (Li and Li, 2006); thereby suggesting a critical role of glia-neuron interactions in the pathogenesis of HD. Further, HD is characterized by the accumulation of mutant Htt in microglia, leading to progressive neurodegeneration through both cell-autonomous and non-cell-autonomous mechanisms (Yang et al., 2017). Microglia as resident immune cells in CNS surveil microenvironment at a quiescent state, which in response to pro-inflammatory stimuli, activates and undergo two separate macrophages (M1 and M2) to release pro-inflammatory cytokines, interleukin (IL)-1b, IL-12, tumor necrosis factor (TNF)-a; and anti-inflammatory cytokines, IL-4, IL-10, and growth factors, transforming growth factor (TGF)-b, mannose receptor (CD206), Arginase1, respectively. Overactivated microglia becomes neurotoxic as they release toxins; hence, the decreasing number of reactive microglia coupled with downregulation of inflammatory cytokines is considered as an indicator of pathologic alleviation of HD (Yang et al., 2017). Thus, therapeutic methods aimed at lowering of neuronal mutant Htt expression along with amelioration of concomitant microglia activation. Several transgenic mice models selectively expressing mutant variants in both neuronal and non-neuronal (astrocytes, oligodendrocytes) cells further provide evidence in this regard. Multiple signaling pathways involving several receptor systems are also crucial in the development of HD pathogenesis such as nuclear factor kappa B (NF-kb) pathway, kynurenine pathway, cannabinoid receptor pathway, N-Methyl-D-aspartate (NMDA) receptor, surface receptors (CD16, CD32, CD68, CD86), toll-like receptors (TLR)-2, -3 and -4; and nonNMDA receptor, a-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) (Yang et al., 2017; Li and Li, 2006). Studies also report the essentiality of wild-type Htt for normal embryonic development, as engineered knockout mutations that disrupt exon 4 (Duyao et al., 1995), exon 5 (Nasir et al., 1995), or the promoter (Zeitlin et al., 1995) of mice HD gene homolog (Hdh), causes its complete inactivation leading to embryonic lethality. Mutant Htt compensates for the absence of endogenous Htt and rescues embryonic lethality of mice homozygous for a targeted disruption of the endogenous Hdh gene (Leavitt et al., 2001); thereby suggesting Htt’s role in embryonic development is independent of polyglutamine tail length. Moreover, HD mutation does not affect the developmental functions of normal Htt, as HD patients appear to develop normally, and the onset of the disease occurs in between 35 and 50 years, which is invariably fatal 15e20 years after the onset of first symptoms. Htt might be required throughout the life, as conditional Hdh knockout mice become sterile and develop a progressive motor phenotype with reduced life span (Dragatsis et al., 2000). However, in spite of these reports, till date, the actual role of normal Htt during adulthood is under debate.

14.2.4 Amylotrophic Lateral Sclerosis (ALS) A meta-analyses reveal 4.48 per 100,000 is the worldwide median prevalence of ALS, and a standardized incidence rate of 1.68 per 100,000 person-years that varied with geography, sex, and age (Logroscino et al., 2018). ALS is rare before 50 years of age, with peak incidence at 70 years followed by a sharp decrease in incidence (Logroscino et al., 2018). ALS is characterized by progressive degeneration of the upper motor neurons in motor cortex and the lower motor neurons in brain stem and spinal cord, death of which causes spasticity, weakness, muscular atrophy, leading to paralysis and death, with a median survival of 3e5 years. ALS shares genetic and pathological features of other ND, such as frontotemporal dementia (FTD) with cognitive impairments, which consider ALS and FTD as the end of the same spectrum of disease (Leblond et al., 2014). Although the majority of ALS occur sporadically, 10% of cases are familial, mainly in an autosomal dominant fashion (Marangi and Traynor, 2014). Genetic contributions to ALS represent the risk of variants of multiple genes that act independently to cause ALS (van Blitterswijk et al., 2012). Out of 50 potential causative genes, superoxide dismutase 1 (SOD1: sequence variants in copper/zinc SOD gene on chromosome 21q12.1, alter mitochondrial oxidative stress mechanism), chromosome 9 open reading frame 72 (C90RF72: GGGGCC hexanucleotide repeat in the first intron of the gene encodes a protein of unknown function on chromosome 9), transactive response DNA binding protein 43 (TARDPB: variant responsible for neuronal cytoplasmic inclusions, immunoreactive for ubiquitin) and fused in sarcoma (FUS: variant alters DNA and RNA metabolism, RNA transcription, splicing, etc.) genes are responsible for >50% of ALS cases (Rosen et al., 1993; Majounie et al., 2012; Neumann et al., 2006, 2009; Chen-Plotkin et al., 2010; Hewitt et al., 2010). Also, vehicle-associated membrane protein B (VAPB), dynactin, valosin-containing protein (VCP), ubiquitin 2 (UBQLN2) and intermediate length expansion of the CAG repeat in ataxin-2 gene (ATXN2) are the rarer genetic causes of ALS; whereas heavy chain neurofilament (NEFH) and progranulin (GRN) are recognized as risk factors for the disease (Morgan and Orrell, 2016); thereby suggesting an enormous role of next-generation sequencing in the pathogenesis of ALS. The pathological hallmarks of

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these adult-onset genes in the post-mortem brain and spinal cord includes abnormal protein misfolding and aggregates in neurons and glial cells although DNA methylation patterns need confirmation. Mutation of the coiled-coil-helix-coiledcoil-helix domain (CHCHD)-10 gene is also identified in ALS patients like, PD and FTD (Imai et al., 2019). In situ hybridization studies of ALS patients show the presence of nuclear RNA aggregates in both cerebral cortex and spinal cord that target to toxic gain-of-function mechanism, whereas the low level of C90RF72 mRNA suggests mutant allele might not be able to produce mature RNA, which points to the hypothesis of loss-of-function process (De Jesus-Hernandez et al., 2011; Gijselinck et al., 2012). Protein synthesis machinery of cell, along with ubiquitin-proteasome and autophagy-lysosome systems, is primarily involved in ALS. Oxidative stress induces ER response, characterized by chromatolysis (i.e., fragmentation and subsequent cytoplasmic dissolution of ER cisternae) and accumulation of aberrant, misfolded proteins in cytoplasmic inclusions are observed in patients with ALS and in experimental models (Kusaka et al., 1988; Oyanagi et al., 2008; Sasaki, 2010). An increase in nucleolar diameter in the transgenic model of ALS linked with cellular attempt to escalate ribosomal gene synthesis in response to stress is also reported (Riancho et al., 2014). Protein aggregates in ALS not only disturb protein homeostasis (proteostasis) and induce cellular stress but also sequester RNA and other proteins, essential for normal cellular functions such as, ubiquitin-dependent degradation, which is manifested by impairment of axonal transport, hyperexcitability, fasciculations, and differential vulnerability of motor neurons (Le Masson et al., 2014). Although preferential factors for the involvement of motor neurons are not fully known, assumptions are made on the following grounds such as, large size and robust cytoskeleton with high metabolic demand for maintaining cell function, a requirement of optimal mitochondrial functions, vulnerability to glutamate excitotoxicity, and dysregulation of intracellular calcium signaling, reduced capacity of heat shock response, chaperone activity, and the ubiquitin-proteasome system (Ferraiuolo et al., 2011; Pasinelli and Brown, 2006). Mitochondrial dysfunction, high level of ROS, ER stress, alteration of ATP, and calcium homeostasis, along with an accumulation of SOD1 (cytoplasmic protein differ from mitochondrial SOD2) in the inner membrane of mitochondria, are also found in ALS patients, as well as SOD1(G93A) mice model of ALS (Tafuri et al., 2015; Bonafede and Mariotti, 2017; Tadic et al., 2014). Neuroinflammatory responses like, activated microglia and lymphocytic infiltrates in spinal cord sections (Henkel et al., 2004), high level of IL-8 in cerebrospinal fluid (CSF) (Kuhle et al., 2009), presence of inflammatory mediators from astrocytes (prostaglandin E2, leukotriene B4, nitric oxides) (Hensley et al., 2006) are contributing to progressive degeneration and phenotypic alteration of motor neurons as the first manifestation of ALS. The overall pathological hallmarks of AD, PD, HD, and ALS in the adult brain especially, motor neurons are summarized below (Fig. 14.1).

14.3 Drug Targets of Protein Aggregates in Neurodegenerative Diseases with Translational Impacts Protein aggregation due to misfolding into toxic forms is the biggest issue in the development of ND, such as, AD, PD, ALS, and HD (Table 14.2). Formation of misfolded proteins due to improper biogenesis, mutations, and physiological stressors simultaneously with unfolded, partially folded, and correctly folded species are natural phenomena in cells (Hartl and Hayer-Hartl, 2009). However, in healthy cells, they are either degraded, or correctly refolded, or sequestered in specific intracellular compartments by chaperone proteins as discussed above. In fact, some protein misfolding creates insoluble amyloid fibrils under certain biochemical conditions (Guijarro et al., 1998; Tzotzos et al., 2010). Many disease-associated amyloidogenic proteins exhibit short, internal amino acid sequences that are necessary for protein aggregation. Once aggregated, due to the thermodynamic stability, they might escape normal degradation process (Knowles et al., 2014). Thermodynamic stability of protein aggregates also contributes to their higher affinity toward native protein aggregation. Cellular aging, disease-associated mutations, etc. hasten up such kind of aggregation ranges from oligomers, amorphous assemblies to highly ordered amyloid fibrils and plaques. Hence, the translational approach of a recent genetically engineered animal model targeting protein misfolding might encounter the dynamic nature of the protein species itself, uncertainty of protein forms like, monomers/oligomers/insoluble aggregates liable to cellular toxicity, as well as interaction between these species. Our limited knowledge and lack of well-validated biomarkers further increase the hurdles in the path of drug discovery. Synaptic dysfunction, improper calcium signaling, mitochondrial abnormality, oxidative stress, and neuroinflammation are the common symbols of ND as discussed here, which suggest bidirectional and mutually exacerbating relation between aggregation of misfolded proteins in AD, PD, ALS, and HD (Taylor et al., 2011; Hohn et al., 2016) (Table 14.2). For example, in AD, pro-inflammatory effects of amyloid b-plaques have a short-term beneficial role but finally cause

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FIGURE 14.1 Molecular events during the pathogenesis of AD, PD, ALS, and HD. Mutation of specific genes and subsequent protein aggregates in a specific subset of motor neurons by oxidative deregulation of mitochondria causes neurodegenerative cell death in the progression of ND, where astrocytes and microglial activation, neuroinflammation, apoptosis, and autophagy play crucial roles.

TABLE 14.2 Protein aggregation in ND and impact on cell physiology with drug targets. ND

Protein aggregation

Cellular physiology

Possible drug targets

AD

Amyloid b-peptide; tau

Tau aggregation inhibitor; Phospho-tau vaccine

PD

a-Synuclein

HD

Htt with tandem CAG (glutamine) repeats in IT15 gene

Acute oxidative stress in neurons, astrocytes and microglia; Impair astroglial antioxidant response; Mitochondrial dysfunction; Increase protein misfolding and aggregation; Neuroinflammation

ALS

SOD1; TARDBP/FUS

Mitochondrial dysfunction; Oxidative stress; Neurofilament accumulation

HSP activation; MSC/ASC and exosomes; Glutamate release inhibitor

Adenosine A2A receptor antagonist Glial cell precursor (use of human/mouse chimera)

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functional impairment of microglia, astrocytes, etc., including their disposal ability of misfolded proteins (Ben et al., 2015; Heppner et al., 2015; Chakrabarty et al., 2015; Guillot-Sestier et al., 2015). Chaperones that help for protein folding, trafficking, and stabilization are another possible therapeutic target with translational importance. Cell type-specific expression of chaperone subsets might explain cell-specific toxicity of misfolded proteins, and thus, guides appropriate chaperones models in this respect. However, a huge number and diversity of mechanism of chaperone pathways, as well as state of the target proteins (i.e., soluble/fibrillar/posttranslational modification, etc.) poses a great challenge to drug development of real translational value, where our knowledge is limited, although there is a possibility of chaperone mutations in cases like, HSP70 and HSP40 mutations in PD (Vilarino-Guell et al., 2014; Wadhwa et al., 2015), VCP mutation in ALS (Johnson et al., 2010), and so on. Further, more than 20 different chaperones overexpressing in cells/animal models of HD, PD, and AD, play critical role in neuroprotection (Smith et al., 2015), thereby suggesting the development of small molecule inhibitors or activators (drugs) of specific chaperones with the opposite effect on target protein stability, aggregation, degradation, and ubiquitination, as well as cell toxicity like, HSP70 and HSP90. The activity of HSP70 and HSP90 are linked via heat shock factor 1 (HSF1), the transcriptional activator of body’s heat shock response. HSF1 is activated by HSP90 inhibitors, which, in turn, induces HSP70 (Thirstrup et al., 2016; Smith et al., 2015; Pratt et al., 2015; Bose and Cho, 2017). Heat shock response diminishes with age and in ND. Studies proved high degradation of HSF1 in mouse and human a-synucleinopathy (Kim et al., 2016), whereas overexpression reduces polyglutamine aggregation and increases lifespan in HD mouse model (Hayashida et al., 2010). Small molecule activators of HSF1 might be neuroprotective in HD model by exacerbating the mutant Htt inclusion body (IB) formation (Bersuker et al., 2013). In contrast, under normal physiological conditions, heat shock response activates only transiently, and multiple cellular mechanisms control these responses (Lamech and Haynes, 2015). However, HSP90 inhibitor in HD mice induces heat shock response and provides short-term beneficial effects (Labbadia et al., 2011). HSP104 shows promising effects in dissolving protein aggregates in AD and PD models (DeSantis et al., 2012). Many other gene mutations in familial PD are also important in this regard (Ciechanover and Kwon, 2015). But as HSF1 promotes tumorigenesis and activates broad-range malignant cancers in humans (Mendillo et al., 2012; Jaeger et al., 2016), so the effectiveness of HSF1 in ND models need to be checked with utmost caution. Animal models and human genetic studies also support the strong association of mitochondrial dysfunction such as, oxidative damage, defective ATP synthesis, NADþ depletion, limited mitochondrial dynamics, and quality control, disrupted calcium homeostasis with protein misfolding in PD, AD, ALS, and HD (Dawson and Dawson, 2017; Kumar and Smith, 2015; Chaturvedi and Beal, 2013). However, whether it is a cause or consequence or part of a self-sustaining mechanism is yet to be decided, but does not create hindrance to therapeutic development for ND. Mitochondria-targeted antioxidants (Co-enzyme Q10, mitoquinone mesylate or MitoQ) and peptide (Bendavia SS31) (Miquel et al., 2014; McManus et al., 2011), transcription factors that increase mitochondrial biogenesis (peroxisome proliferator-activated receptor gamma coactivator 1a, or PGC1a; nuclear factor erythroid 2-related factor 2, or Nrf2; AMP-activated protein kinase, or AMPK) (Johri and Beal, 2012), and molecules that replenish NADþ levels (nicotinamide mononucleotide, NMN) are beneficiary in animal models (Bido et al., 2017; Long et al., 2015; Wang et al., 2017). However, these are limited to clinical trials only, might be due to too late entry of the patient for treatment when pathophysiology of ND (i.e., symptomatic stage) becomes too late to intervene at the mitochondrial level. In contrast, treatment targeting mitochondrial dysfunction starts before the onset of the clinical symptoms in preclinical animal models. Moreover, in reality, once cell damage starts, other factors like, inflammation and vascular damage in ND come forward, leading to neuronal cell death response. Antibody targeting of misfolded proteins in ND, especially AD, has a good future as they cross blood-brain-barrier (BBB) to bind with amyloid b-plaque in extracellular space (Wisniewski and Goni, 2015). Most of the misfolded proteins of ND discussed here, exhibit extracellular leakage, for which highly selective monoclonal antibodies, at least, in theoretically, are available (DeGenst et al., 2014; Vaikath et al., 2015). However, nonspecific engagement and uncertainty of clinical forms of plaques in the patients make the situation more critical while considering their translational values. Mammalian target of rapamycin (mTOR)-dependent inhibitors of autophagy, including natural substance curcumin and resveratrol, are entering clinical trials for treating ND. But mTOR regulates many cellular processes in addition to protein degradation. Also, mTOR inhibitors cause undesirable side effects in clinical trials although they may endorse large-scale balancing of protein synthesis and degradation in the cellular system. Molecular chaperones, HSP70 and HSP40 promote folding of Htt into a native structure, and facilitate the recognition of abnormal proteins, promoting either their refolding, or ubiquitination, and subsequent degradation by 26S proteasome (Lackie et al., 2017). HD mutation induces conformational changes of the protein (i.e., Htt misfolding), which, if not corrected by chaperones, accumulates into the cytoplasm (Finkbeiner, 2011), and elicit toxicity by either full-length, or cleaved mutant Htt that form soluble monomers/oligomers, or large insoluble aggregates, and impair ubiquitin-proteasome

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system, resulting in more protein aggregation leading to impairment of vesicular transport and clathrin-mediated endocytosis (Finkbeiner, 2011; Hunter et al., 2007). Mutant Htt in the mitochondrial outer membrane causes impaired electron transport chain (ETC) complexes II and III with concomitant depletion of intracellular ATP pool, inhibition of TCA cycle, and increase ROS levels (Mochel and Haller, 2011). Further, reducing GTPase dynamin-related protein 1 (DRP1) activity restores early mitochondrial fragmentation and transport, and improves phenotype in HD mice (Song et al., 2011), suggesting the significance of perturbed mitochondrial function in HD and potentiality of mutant variant in neuronal dysfunction by disrupting energy metabolism and promoting oxidative damage. The involvement of SIRT1 in the regulation of HSF1 by enhancing DNA binding activity is of utmost interest to find whether increase HSF1 contributes to SIRT1-mediated neuroprotection in HD mice (Westerheide et al., 2009). Also, mutant Htt could activate proapoptotic proteins directly, or indirectly by mitochondrial damage, leading to greater cellular toxicity, translocation into the nucleus to form nuclear inclusions, which disrupt chromatin structure, block histone deacetyltransferases, and hence, the transcription process, as well as ubiquitin-proteasome system in the neuron (Gray, 2011; Anglada-Huguet et al., 2017). Based on these observations, several inhibitors of histone deacetyltransferases in different animal models of HD report cell survival, reduce brain atrophy, restore neurotransmitter inputs, rescue mutant Htt toxicity and memory deficit, as well as improve proteasome mechanism and motor neuron performance (Anglada-Huguet et al., 2017), which definitely show rays of hope for clinical trials, a future path to cure HD. Currently no treatment available that can halt ALS progression; but advancement in genetic sequencing, biochemical techniques, experimental models, and imaging system open new avenues for drug development highlighting RNA biology and protein degradation, specifically after the discovery of FUS and C90RF72 genes. Animal and human studies reveal critical implications of specific neuromuscular junctions in the initial phase of ALS, but despite efforts, drug targets are challenging due to overlapping of similar clinical features of other disorders. However, the combination of specific parameters of such junctions and laboratory tests are crucial for early determination of changes in muscles, motor neuron, and the prediction of ALS progression, which might have translational importance to discriminate ALS and ALS-mimicking situations (Campanari et al., 2019). Further, the trials of glutamate release inhibitor riluzole (targeting glutamate excitotoxicity), Nogo-A monoclonal antibody (encouraging nerve growth), animal model demonstrating axoplasmic flow (for long axon of spinal nerves), phase I study of intrathecal antisense oligo, ISIS333611 (on SOD1 mutations), and ATXN2 (for ataxin), are continuing, although conclusive remarks on ALS treatment are yet to come (Morgan and Orrell, 2016). Bonafede and Mariotti (2017) proposed mesenchymal stem cells (MSC) as a promising candidate to treat ALS, because of the fact that MSC support motor neurons and surrounding cells, reduce inflammation, stimulate tissue regeneration and release growth factors, the requisite for altering the condition of ALS. Further, as adipose tissues are easily available from liposuction, autologous transplantation of adipose tissue-derived MSC (ASC) gains the attention of the scientists to treat ALS (Bonafede and Mariotti, 2017). The beneficial effects of ASC might be due to paracrine activity rather than engraftment, which suggests that exosomes or extracellular vesicles and microvesicles that transfer long-distance signals, might be a better approach of non-cell based therapy for the treatment of ALS (Bonafede and Mariotti, 2017). As these vesicles preserve immune properties of their origin and released by MSC to recapitulate the effect of stem cells, they could further strengthen their transfer to ALS patients without immunogenic reactions. Also, research is going on to identify downstream targets of any genes/gene products linked with ALS, as discussed above, and to find a universal therapy not only for ALS but also for other ND. Overall, although molecular network of misfolded protein (i.e., protein aggregates) is a challenging area of drug development with translational future to treat AD, PD, ALS, and HD using genetically engineered animal models, but very few of them, till date, reveal significant benefits in clinical trials, which might be due to cytotoxic profile and reduce BBB penetrating activity of the respective target molecules (Pratt et al., 2015; Fontaine et al., 2016; Wang et al., 2016).

14.4 Conclusion and Future Direction of Research Overall, the primary goal of the development of animal models is to identify the key points of neurodegeneration that represent therapeutic targets. Till date, animal models provide remarkable contributions to understand the pathogenesis and molecular mapping of ND such as, AD, PD, HD, and ALS. However, in most of the cases, clinical trials based on the success of these animal models fail to slow down the degenerative changes, might be attributed to numerous factors, including designing issue and optimistic interpretation of the preclinical study, unavailability of 100%-matched animal models, lack of target number of human engagement in the clinical trial, and absence of informative biomarkers from clinical sides. Also, as these diseases in human are heterogeneous i.e., both pathological and clinical/behavioral domains are present; hence, animal models, in reality, recapitulate only a part of this complex scenario. Further, attention is focused

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mostly on transgenic models, but in humans, these diseases especially, AD and PD, are sporadic rather than familial (Ribeiro et al., 2013). This causes an enormous challenge for the development of next-generation animal models for the treatment of such ND, and more in-depth breakthrough is still required to elucidate the minute details of the relation between biological networks and progression of ND. Even in multiple models, thorough preclinical/experimental studies emphasized on both potentiality and limitations of a particular intervention instead of concentrating on one model at one-time point, are highly essential. Today, treatment strategy also requires simultaneous targeting of multiple components, including some disease-specific domain(s) for assessing or modulating neurodegeneration. At present, molecular mapping of drug targets with translational values is most important in animal biotechnology, as well as pharmaceutical sciences, which involve several stakeholders such as, academia, industry, private foundations, Govt. funding agencies, patients and caregivers, and policymakers. The team approach is really critical for proteinopathies, which might be due to heterogeneity and/or rarity of the conditions, and difficulty of recruiting sufficiently large patient cohorts for clinical trials for ND with genetic predisposition like, AD, PD, HD, and ALS. In preclinical models, although in most of the cases, scientists tried to mimic the human disease scenario, sometimes it induces autoimmune response during clinical trials (Cribbs, 2010; Wisniewski and Konietzko, 2008). Moreover, neurodegenerative animal models (mice) are sometimes challenged because ND in patients does not occur naturally in experimental studies due to species differences and aging components that take decades to manifest in humans, although >95% of genes are conserved among them. Further, the mouse is used as an assay system rather not to reflect the entire disease population. Hence, for a proper understanding of the differences between these ND in patients and accurate interpretation of the experimental results are essential. Practically, till date, the mouse model is relevant and the best known mammalian model for studying human disease at phenotypic, molecular, and clinical levels (Perlman, 2016). Also, advances in genetic engineering in mice open quick and efficient modeling of allelic variations, and enabling us better model for ND. Hence, despite hurdles mentionedabove, considering the increasing human burden, loss of quality of life, and extreme value of experimental animal models tested so far, it is not justified to accept defeat in research efforts to address these age-old genetic ND. Animal models provide valuable investigation platform for biological processes, including molecular signaling that occurs in vivo in the context of amyloid-b or tau (AD), a-synuclein (PD), Htt (HD) and SOD1 (ALS), which might serve as potential pharmacological biomarkers (i.e., possible drug targets). One of the better strategies might be targeted pathway(s) that increase the vulnerability of neurons to excitotoxicity (Mattson, 2003). Glutamate excitotoxicity and calcium homeostasis are the important drug targets in this regard by modulating neurotrophic factor signaling cascades and stress response pathways, which guard neural tissues under normal circumstances. Glial cells that express glutamate transporters, cytokines, and specific glutamate receptor antagonists might be considered as potential neuroprotective agents in CNS, and one of the important drug targets of ND so far as the motor neuron vulnerability to excitotoxicity is concerned. Second, balancing the proteostasis of protein aggregates such as, amyloid-b/tau, a-synuclein, or Htt by enzymatic corrections, as well as the application of specific chaperones/activators with higher BBB crossing ability holds novel possibilities for future treatment of AD, PD, and HD, respectively. The role of other lysosomal enzymes as possible risk factors for ND should be taken into account in this kind of endeavor, which might provide an insight into the mechanism of synaptic plasticity. Third, molecular docking and in silico approaches with special emphasis on dysregulated chaperone are also important future perspectives of research in management of these ND, which could elucidate the underlying mechanism of proteinopathies, and generate effective and unambiguous pharmacological therapies. Fourth, small-molecule therapies targeting altered mitochondrial biogenesis in AD, PD, HD, and ALS are another emerging area to treat common pathologies of ND and have translational prospect, particularly those to be designed to prevent mitochondrial damage, increase organelle biogenesis, or enhance mitochondrial quality control. Last, but not least, considering multiple pathways and complex molecular network, biomarkers and treatment strategies might require simultaneous targeting of multifactorial therapeutic approaches, including wide-spectrum laboratory analysis with the highest translational impact for treatment of ND even where a single gene mutation (like, HD) is involved. Therefore, the development of clinically relevant biomarkers and improvement of trial design are of utmost medical importance for faster evaluation of next-generation drugs to reduce the burden of AD, PD, ALS, and HD in the society.

Acknowledgment Author grateful to Ms. Vishakha Shrimali, Ph.D. student, for cross-checking the references, and Dr. Arnab Banerjee, BITS Pilani-Goa for guidance in plagiarism checking. Article preparation has been partially supported by DST-SERB project (EMR/2017/004150), Govt. of India.

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Conflict of Interest None.

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Chapter 15

Issues and policies in animal genomics Ramanuj Banerjee1 and Sukanta Mondal2 1

Department of Scientific & Industrial Research, Ministry of Science & Technology, Government of India, New Delhi, Delhi, India; 2Principal

Scientist, ICAR-National Institute of Animal Nutrition and Physiology, Adugodi, Bangalore, Karnataka, India

15.1 Introduction In the modern era, the technological applications of biological processes are broadly defined as “Biotechnology.” The new technological processes are explored in living organisms and/or their body parts by biotechnologists for the development of new products or processes, which can improve the quality of life and/or environment. In agriculture, medicine, energy, industry, and environment sectors, the various applications of biotechnology and its tool, processes are implemented. The leading arenas of today’s biotechnology refer to genetic engineering comprising of genomics, recombinant DNA (rDNA) technology, proteomics, and bioinformatics, etc. Animal genomics precisely leads to the analytics of genetic architecture in respect to the elucidation of genes of economic interest. Apart from rDNA, other technologies have emerged that are intended to alter the genomes of various organisms, including animals. Some of those technologies include the use of “nucleases” or “genome editing technologies,” including engineered nuclease/nucleotide complexes such as zinc finger nucleases (ZFN), transcription activator-like effector nucleases (TALENs), and the clustered regulatory interspersed short palindromic repeats (CRISPR) associated systems. Those nucleases are defined to introduce alterations at specific sites in the genome, rather than the more random changes associated with rDNA technology. In present time, the application of various biotechnological strategies and tools is implied in healthcare, agriculture, veterinary science, microbiology, social wellbeing, and environmental management industries for high returns with a greater impact on the global economy. Genetic Engineering is such a powerful biotechnological tool, which creates environment-friendly alternatives for products and processes. Those alternatives result in an overwhelming diversity of species, biomolecules, and metabolic pathways on this planet. The alternative products and processes also diminish environmental pollution and generate nonrenewable resources as byproducts. In present days, additional genetic properties can be incorporated to supplement naturally occurring organisms for biodegradation of specific pollutants if it is not possible to degrade properly or quickly by naturally occurring organisms. It is noted that in combining different metabolic abilities within the same microorganism, the environmental cleanup process may be circumvented and exaggerated. In biopharmaceutical research, biotechnical methods are used to produce several recombinant proteins for various applications. The first genetically engineered product, “Human insulin,” was produced commercially (1982) from nonvirulent strain of Escherichia coli bacteria by the introduction of a copy of the gene for human insulin. When the gene was amplified, the bacterial cells produced large quantities of human insulin that were purified and used to treat diabetes in human beings. Axnumber of other genetically engineered products have been approved since then, including human growth hormone, alpha interferon, recombinant erythropoietin, and tissue plasminogen activator, etc. Biotechnology techniques are also being applied to plants to produce plant materials with improved composition, functional characteristics. The plant materials with improved composition and functional characteristics are also produced by biotechnology techniques or genetic engineering tools. The slow-ripening tomato was the first commercially available whole food product. The gene for the enzyme polygalacturonase responsible for softening is turned off in this tomato. The disease-resistant, pests-resistant and environmental condition-resistant plants are also being developed. In 1995, the Environmental Protection Agency (EPA) issued permission for the development of transgenic corn seed, cottonseed, and seed potatoes that contain genetic material to resist certain insects. Such genetic materials are claimed to have the properties of environmentally friendly herbicides and pesticides having less toxicity. The use of recombinant bovine somatotropin (BST) in dairy cows was the first

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approved application of biotechnology techniques or genetic engineering tools in animal production. Bovine somatotropin (a protein hormone) is found naturally in cows, which is necessary for milk production. Milk production has been shown to increase by 10%e25% with the administration of recombinant BST in dairy cows under ideal management conditions. In animal production and animal health, biotechnological techniques or genetic engineering tools are used for the development of artificial insemination, animal cloning, animal vaccines, and rapid disease detection kits and also for improvement of growth rate and/or feed efficiency, etc.

15.1.1 Genomic technologies in animal husbandry In the present context of genetic engineering and related technology, there are enormous applications for animal husbandry with respect to companion animals, wild animals, farm animals, and animal models used in scientific research. The genetically engineered animals are mostly in the research phase. In the future, their actual commercial use is expected toward intended applications. Companion animals: “GloFish has been developed by inserting genes from sea anemone and jellyfish into zebrafish with the help of genetically engineering. It is for expression of fluorescent proteins. GloFish began to be marketed in the United States in 2003 as ornamental pet fish. However, their sale made controversial ethical issues in California and to prohibit the sale of ‘GloFish’ as pets. Insertion of foreign genes, gene knock-out techniques are also being used to create designer companion animals. As an example, creation of hypoallergenic cats some companies removed the gene that codes for the major cat allergen Fel d1 through genetic engineering technology. Companion species was derived by cloning. During 2002, first cloned cat was created which was nomenclature as ‘CC,’”. After few years, first cloned dog, “Snuppy” was created. However, apart from some isolated cases, the genetically engineered pet industry is yet to move forward. Wild animals: Genetic engineering to wild species mainly involves cloning. It applies to either extinct or endangered species like cloning of the extinct thylacine and the woolly mammoth. Farm animals: Genetic engineering applies in farm animals toward improving animal productivity, food quality, disease resistance, and environmental sustainability. As an example, transgenic pigs and sheep that have been genetically altered to express higher levels of growth hormone. For enhancing food quality, pigs have been genetically engineered to express the D12 fatty acid desaturase gene (from spinach) for higher levels of omega-3, and goats have been genetically engineered to express human lysozyme in their milk. Those developments may add nutritional value of animal-based products. For creating disease-resistant animals genetic engineering technique conferred immunity to offspring via antibody expression in the milk of the mother, disruption of the virus entry mechanism for pseudorabies, resistance to prion diseases, parasite control in sheep, and mastitis resistance in cattle, etc. It also aims to reduce agricultural pollution. The best-known example is the Enviropig, a genetically engineered pig having ability to produce an enzyme that breaks down dietary phosphorus (phytase), and thus restricts the phosphorus release quantity in its manure. There is lot of debate and resistance to the commercialization of genetically engineered animals like AquAdvantage Atlantic salmon for food production and commercial application. Scientists have made an effort to generate genetically engineered farm species such as cows, goats, and sheep that express medically important proteins, therapeutic recombinant proteins in their milk. The first therapeutic protein ATryn was developed through genetically engineered animals to be approved by the Food and Drug Administration (FDA) of the United States. It is used as a prophylactic treatment for patients having hereditary antithrombin deficiency and are undergoing surgical procedures. Research animals: Genetically engineered animals for biomedical applications are of numerous. It includes gene function, modeling of human disease to either understand disease mechanisms or to aid drug development, and xenotransplantation. With the process of addition, removal, or alteration of genes, scientists can pinpoint the effect of that particular gene in biological systems in respect of deviated structure, different or altered function. Genetic engineering enables to develop human disease models for understanding how and why a particular disease develops, and what can be done to halt or reverse the process such as Alzheimer’s disease, amyotrophic lateral sclerosis (ALS), Parkinson’s disease, and cancer, etc. Application of genetic engineering (e.g., Transgenic and knock out mouse) has immense use in the pharmaceutical industry, especially in drug discovery, drug development, defining potential drug targets, and risk assessment activities. In extensive pharmaceutical research for the screening of drug targets through genotoxicity, carcinogenicity, drug-induced immunotoxicity, immune and inflammatory responses, genetically engineered animal models like the knock out mouse, nude mouse, etc. are used. It is generally accepted that the use of genetically engineered animals in science is mainly related to basic research viz genetic-transplantation of cells, tissues, or whole organs from donor as an animal to the recipient as a human. As an example, scientists have developed a genetically engineered pig aiming at reducing thw rejection of pig organs by humans. It is currently at the basic research stage, but it shows great promise in the organ transplantation process substituting donated organs. All genomics techniques are needed to pass through an

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extensive validation process like phase-wise clinical trials. However, all sorts of animal genomics issues are being addressed through robust global or state policies to safeguard ethical concerns, environmental protection, animal integrity/ dignity, and long-term undesirable effect on society.

15.2 Global (transcontinental) scenario in transgenic animal research: issues and policies 15.2.1 North America Canada: In Canada, genetically engineered animals are defined through various terminology like genetically manipulated, genetically altered, genetically modified, transgenic, and biotechnology-derived, etc. The primary technology used for genetic engineering during the early era was the only transgenesis. It was restricted to the transfer of genetic material between organisms. However, with the advancement of science and technology, recent applications include the creation of genetically engineered animals through manipulation of genes already present or deletion of genes, including insertion or substitution. Accordingly, guidelines developed by the Canadian Council on Animal Care (CCAC) has been revamped with the new arena of the term “genetically engineered.” As per the adoption of the CCAC, the genetically engineered animal is defined as an animal having changes in its nuclear or mitochondrial DNA. The changes include insertion, deletion, addition, and substitution of animal host cell genetic material. The changes also include the alteration of host cell genetic materials with foreign DNA through technological intervention. The animals that have undergone induced mutations by chemicals or radiation or due to any other mutagenic substances, unlike spontaneous natural mutations, occur in populations, and cloned animals are considered as genetically engineered animals since there is direct intervention in the creation of those animals. Since there are direct intervention and planning in the creation of cloned animals, it is considered as genetically engineered products. There are three types like DNA cloning, therapeutic cloning, and reproductive cloning. However, reproductive cloning out of all three types most likely to lead to animal welfare issues. Therefore, during the development of “CCAC guidelines on genetically- engineered animals used in science” key ethical issues with animal welfare concerns were identified and captured as follows: (i) invasiveness of procedures, (ii) large numbers of animals required, (iii) unanticipated welfare concerns, and (iv) how to establish ethical limits to genetic engineering. The CCAC defines accepted ethics of animal use in science. It includes principles of the Three “R” (Reduction in numbers of animals, Revamping of practices and animal husbandry to diminish pain and distress, and Replacement of animals with nonanimal alternatives as much as possible). United States of America (USA): In USA, during Asilomar Conference in February 1975, recognizing concerns related to potential hazards due to emerging gene technologies (like gene transfer, etc.), scientists tried to reach a consensus to self-regulate research involving rDNA technology until its safety could be assured appropriately. The National Institutes of Health (NIH) published research guidelines in 1976 for using rDNA techniques. The primary federal entity, “NIH Recombinant DNA Advisory Committee,” is empowered to review and monitor DNA research till 1984. However, due to a legal challenge forced the US Administration to consider and propose policies to guide activities of federal agencies responsible for reviewing biotechnology research and its products. The White House Office of Science and Technology Policy (OSTP) published a “Coordinated Framework for Regulation of Biotechnology,” during 1984. The framework proposed that genetically engineered products would continue to be regulated as per their characteristics and novel features but not by their method of production. It also proposed that new biotechnology products will be regulated under the existing web of federal statutory authority and regulation. The framework was finalized by OSTP during 1986. The framework identified lead agencies like FDA, APFHS, and EPA to coordinate activities with responsibility. For example, the Food and Drug Administration (FDA) is responsible for regulating food and feeds in the market after modification through genetic engineering. The US Department of Agriculture (USDA), Animal and Plant Health Inspection Service (APHIS) are designated for importation, interstate movement, and environmental release of transgenic plants that contain plant pest components. Through permits, it licenses field-testing of food crops prior to commercial release. Sometimes responsibilities are overlapped as some plants are modified to contain plant pesticides. Before the distribution and sale of certain pesticides produced in transgenic plants, the Environmental Protection Agency (EPA) registers those pesticides. EPA also establishes pesticide tolerances for residues in foods. Therefore, in such cases, APHIS and EPA together established procedures to review and approve field tests of modified plants and microorganisms. To withdraw a food from the market, FDA has postmarket authority. The framework has considered an amendment of regulatory policies to protect public safety and the environment with the development of new technology during allowing genetically modified crop development to progress.

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15.2.2 South America Argentina: Argentina is the world’s second-largest exporter of GM crops. The biosafety system of Argentina is more or less similar to Egypt. In response to domestic interest and research in GM technologies, its national biosafety system was established in 1991. As desired by the USA and transnational seed companies for using Argentina’s field as a location for off-season GM seed production and field trials, Argentina has put its biosafety system in place appropriately.

15.2.3 Australia/Oceania Innovative genomic technology era commenced in Australia during 2001 with the commencement of new commonwealth gene technology act 2000. The Act deals with modified gene technology (GMOs) of any organism, including research, manufacturing, production, and importation. One central, enforceable scheme has been provided by the Act to regulate GMOs. The Act includes the organization structure of an independent statutory officer, the gene technology regulator (GTR). The role of the GTR is to manage GMO licenses, assist in the development of policy principles and guidelines, and provide information and advice about GMOs to other agencies and to the public. Gene technology-related community concern is captured appropriately in the Act through the involvement of three committees; a Technical Advisory Committee, a Community Consultative Committee, and an Ethics Committee. The three committees advise the GTR and the Ministerial Council on the issues related to Gene Technology. The Act has the scope of creating more streamlined pathways for industry and researchers seeking approval.

15.2.4 Europe United Kingdom (UK): In the UK, Genetically Modified Organisms (GMOs) are regulated by a number of stringent regulatory procedures under Genetically Modified Organisms (Deliberate Release) Regulations. Applicants willing to carry out GMO tests have to register with the Health and Safety Executive (HSE). Certain tests are required for detailed notification that is reviewed by HSE as well as the number of Government departments and advisory committees. Smallscale field trials are governed by the EU’s “Deliberate Release” Directive. The detailed application for field tests should be submitted to the Department of the Environment, Transport, and the Regions (DETR). The application is reviewed comprehensively by the Advisory Committee on Releases to the Environment (ACRE). Membership of ACRE includes experts from ecology, biodiversity, and agronomy. For producing and marketing of GM crops at commercial scale, the applications are reviewed both on national level and by the other Members States of the EU. In the UK, applications are reviewed by three committees of Ministry of Agriculture, Fisheries and Food, as well as Advisory Committee on Novel Foods and Processes, and the Committee on Chemical Toxicity of Consumer Products, Food, and the Environment. In respect of the application of particular pesticides applied to a GM crop, separate approval for that pesticide is required by the Advisory Committee on Pesticides, which is a little lengthy process. There is no commercial growing, but there have been experimental trials of GM potatoes, wheat and Camila sativa (“false flax”) during recent years. In 1998, a ministerial group on biotechnology and genetic modification was set up to consider wide range issues arising from genetic modification. The Ministerial group reviewed the regulatory controls and recommended for setting up of two new commissions during 2001. The commissions are named as “Agriculture and Environment Biotechnology Commission (AEBC)” and “Human Genetics Commission (HGC).” AEBC is mandated to work alongside the European Food Standard Agency (EFSA) is actually a strategic advisory body for the safety of GM food. The working of the advisory committee is transparent, reports are published on websites, committee memberships are reviewed, and it includes consumer representation. Russia: In Russia, there are different committees, state divisions/departments are responsible for Regulation on Genetic Engineering, biotechnology products inspection, registration, and release. Inter-Agency Committee on Genetic Engineering Activity (1997) was constituted as a permanent body for necessary coordination and recommendation related to inspection and registration of confined field trials for GM-plants in the different agro-climatic zones of Russia. Department of State Sanitary and Epidemiological Surveillance under the Ministry of Health is responsible for the safety of novel foods and their state registration since 2000. The responsibility regarding Biosafety problems, including GMOs biosafety, and their State registration belongs to the Ministry of Industry, Science and Technologies (Division of the State Regulation on Genetic Engineering, March 2001). Inter-Agency Committee on Biotechnology (2002) was also constituted as a permanent body with coordinating and recommendatory functions in this area. Ministry of Agriculture (Department of Veterinary, since November 2002) is responsible for safety of novel feeds and their State registration. Advisory Council on Biosafety under the Ministry of Agriculture (2002) has a major function of risk assessment of novel feeds before their State

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registration by the Ministry. Between 1996 and 2002, many acts, laws, and decisions of Russian Federation have been enacted/revised to deal with GMOs, novel foods, feeds, diagnostics, and healthcare products. Two GM-potatoes resistant to Colorado beetle (Monsanto) were registered in March 2002 to release into the environment. The Ministry of Health has registered ten genetically modified agricultural cultures like some species of soybeans, potato, and maize for their industrial use and for consumption as food products.

15.2.5 Asia China: Regulation on the control of genetically modified (GM) plants, animals, and microbes aiming to protect human health, ecology, and the environment have been implemented in China with effect from 2001. The regulation consists of 56 articles aiming at strengthening the control over the research and development, production, processing, and trading of genetically modified agricultural products, including plants, animals, and microbes. The regulation includes mandatory assessment on the safety aspects of GM products and its appropriate labeling. As per regulation, research institutions working on GM research and or GM development are required to have sufficient facilities and techniques to ensure the safety of their research and development of GM products. Those institutions should apply to the State Agriculture Administration and Department (SAAD) or Provincial Agriculture Administration and Department (PAAD) or safety certificates to release their final products with appropriate labeling after completion of all scheduled productive experiments. The Agricultural Administration Department of the State Council is responsible for approving the import of GM products and quarantine institutions for inspecting the certifying nonGM products to be exported. The Agriculture Administration Department of the State Council is also authorized to ban the production, processing, and trading of any GM product that is found to have posed a hazard to human health, ecology, and the environment. Japan: In Japan, the scientific and experimental activities are regulated by the Ministry of Education, Culture, Sports, Science and Technology. The Science Committee for Environmental Safety Assessment of GMOs undertakes biosafety reviews and advices the Ministry of Agriculture, Forestry and Fisheries. Ministry of Health, Labor, and Welfare and Ministry of Economy, Trade, and Industry are responsible, respectively, for the development, manufacturing, and industrial use of food additives and pharmaceuticals. Experimental activities are separated between research facilities of universities and others, which are regulated through rDNA guidelines. For commercial application there are separate guidelines for GMOs, GM feed, feed additives, foods, pharmaceuticals, and for industrial use, it is governed by different ministries. Accordingly, depending upon the nature of the rDNA product, applications are essentially required to forward to different concerned departments. Japanese Government has stringent stricture toward the evaluation of the safety parameters of all GMOs, developed by producers and trading companies as per the Government guidelines. Thailand: In Thailand, an autonomous organization, the National Center for Genetic Engineering and Biotechnology (BIOTEC), was established during 1983. This Center was later attached to the National Science and Technology Development Agency in 1991. The BIOTEC established the National Biosafety Committee (NBC) during 1993. NBC introduced biosafety guidelines for laboratory and fieldwork, as well as the release of Genetically Improved Organism (GIO) into the environment. Accordingly, Biosafety committees at various public institutions and private companies were also constituted. However, plant quarantine law executed by the Department of Agriculture, Ministry of Agriculture, regulates the importation of prohibited material. The permission related to field testing of imported transgenic plants is the responsibility of the Ministry of Agriculture. The BIOTEC realizes that genetic engineering related to scientific and nontechnical matters related to biosafety is dependent upon public support and building capacity. Philippines: In Philippines, during 1990 through Executive Order 430, regulations of R&D in biotechnology are made through the creation of a National Committee on Biosafety (NCBP). NCBP is administratively under the Department of Science and Technology, but its members come from other agencies such as the Department of Agriculture, Health, and Environment. The Committee consists of eminent scientists from biology, environment, physics, social sciences, and members from the community, including NGOs. NCBP had developed three Biosafety Guidelines for R&D. Biosafety Guidelines for Small-Scale Laboratory Work; Biosafety Guidelines for Large-Scale Contained Work and Glasshouse Trials; and Biosafety Guidelines for Planned Release of Genetically Modified Organisms (GMOs) and Potentially Harmful Exotic Species (PHES). The R&D proposals for contained or limited field trials of GMOs, both by local or foreign researchers or private sectors, are subjected to the approval through NCBE guidelines. The country has drafted guidelines for the commercialization of GMO plants also. Those guidelines had undergone several nationwide consultations with various stakeholders consisting of farmers, fisherfolks, NGOs, consumer groups, private sector, and academia before approvals from the Secretary of the Department of Agriculture. India: In India, the Environment Protection Act (EPA) 1986 and Rules 1989 regulate all Acts, rules, and regulations, as well as procedures for handling genetically modified organisms (GMOs), and recombinant DNA (rDNA) products. During

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1990, a set of rDNA guidelines were issued covering genetically engineered organisms, genetic transformation of plants and animals, mechanism of implementation of biosafety guidelines, containment facilities under three risk groups covering manufacture, use/import/export, and storage of hazardous microorganisms. During 1993, genetically engineered organisms or cells came into force in India. For collating with the needs of scientific know-how, the guidelines were revised in 1994 as “Revised Guidelines for Safety in Biotechnology.” During 1998, “Revised Guidelines for Research in Transgenic Plants and guidelines for Toxicity and Allergenicity for Evaluation of Transgenic Seeds, plants and plant parts” had come into force to provide a special review for genetically engineered plants. Office of the Drug Controller General of India (DCGI), implement Drugs and Cosmetics act. DCGI also published separate guidelines for clinical trials of recombinant products and ethics. There are several departmental/Ministerial Committees at policy and execution levels. In India, the Department of Biotechnology, Ministry of Science and Technology, Govt. of India, provides recognition to Institutional Biosafety Committees (IBSC) and also services a Review Committee on Genetic Manipulation (RCGM) for regulating research and limited field experiments. On the recommendations of the RCGM, the Genetic Engineering Approval Committee (GEAC) of the Ministry of Environment and Forests provides clearance from the safety angle for commercial purposes. The Ministry of Health and Family Welfare (MoHFW) provides final licenses for recombinant products of healthcare in accordance with the Drugs and Cosmetics act implemented by the office of the Drug Controller General of India (DCGI) in association with Central Drugs Standard Control Organization (CDSCO). Separate guidelines for clinical trials of recombinant products and ethics are also published by MoHFW. In addition, biosafety measures during field trials, including Biosafety Research Level I (BRL I) and Biosafety Research Level II (BRL II) in the case of GE plants (DBT and MoEFCC, 2008a,b), have been captured in “Revised Guidelines for Research in Transgenic Plants.” The BRL1 and BRL II have also been mentioned in the “Guidelines for Toxicity and Allergenicity Evaluation of Transgenic Seeds, Plants and Plant Parts, 1998.” Both guidelines are published by DBT, MoST, Govt.of India. Protocols for Food and Feed Safety Assessment of GE crops published by DBT during 2008 captured Study regarding acute Oral Safety Limit in Rats or Mice, in Rodents Subchronic Feeding Study, Protein Thermal Stability study, Pepsin Digestibility Assay, and study on livestock feeding, etc. A systematic approach to analyze the risk and safety of foods developed through GE plants on human health are described in the Guidelines for the Safety Assessment of Foods Derived from Genetically Engineered Plants published by ICMR, New Delhi on 2008 (ICMR, 2008). Therefore, as cumulative, in order to Evaluate Proposals, Government of India [Ministry of Environment, Forest and Climate Change (MoEF&CC), Department of Biotechnology under Ministry of Science and Technology (DBT, MoST), Ministry of Health and Family Welfare (MoHFW), Ministry of Agriculture and Farmers Welfare (MoAFW), National Biodiversity Authority (NBA) Food Safety and Standards Authority of India (FSSAI) has issued following guidelines: I. II. III. IV. V. VI. VII. VIII. IX. X. XI.

The Environment Protection Act (EPA) 1986 and Rules 1989 (MoEF&CC) Recombinant DNA Safety Guidelines, 1990. (DBT, MoST) Recombinant DNA Safety Guidelines and Regulations, 1994. (DBT, MoST) Revised Guidelines for Safety in Biotechnology (1994). (DBT, MoST) Revised Guidelines for Research in Transgenic Plants (1998). (DBT, MoST) Guidelines to generate preclinical and clinical data for recombinant DNA (rDNA) Vaccines, diagnostics, and other Biologicals, 1999. (DBT, MoST) Regulations and Guidelines for Recombinant DNA Research and Biocontainment, 2017 (DBT, MoST) Drugs and Cosmetics Act, 2018 (MoHFW) In addition to the above Acts and Rules, other acts/orders are related to GMOs are as follows: Plant Quarantine Order, 2003. (MoAFW) Biological Diversity Act, 2002. (NBA) Food Safety and Standards Act, 2006. (FSSAI)

15.2.6 Africa Egypt: During 1995, through a ministerial decision, Egyptian National Biosafety Committee (NBC) was established for putting together policies and procedure to govern the use of biotechnology in the country. Before that, Egypt’s regulation did not include guidelines for handling transgenic materials under contained conditions, nor did they cover the release of Genetically Modified Organisms (GMOs) into the environment. The Agricultural Genetic Research Institute (IGRI) prepared the regulations and guidelines, which was revised by NBC and was approved by another ministerial discussion during 1995. Under the NBC, Institutional Biosafety Committee (IBSC) has been constituted for implementing the biosafety guidelines and suggesting additional procedures, inclusions from time to time. The Supreme Committee for food

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safety under the Ministry of Health and Seed Registration Committee under the Ministry of Agriculture acted upon commercialization and monitoring as per the proceedings of NBC. However, only the Minister possesses the power of granting official approval. The Ministry of Environment has limited involvement in the whole regulatory process.

15.2.7 Antarctica It is noted that the biota of the Antarctic is highly endemic. The abundance of taxonomic groups and their diversity differs from elsewhere in the world. Such differential characteristics from other parts of the globe have resulted from evolution in isolation in an increasingly extreme environment over the million years. Antarctic species represent the best examples of natural selection at the molecular, structural, and physiological levels. Geological and climatic cooling events over the period influence molecular genetics data to be consistent with the diversity and distribution of marine and terrestrial taxa. Both in the past and the present day, limited habitat availability has played a major role in structuring populations of species. Populations of species are often geographically structured into genetically distinct lineages, due to above geographical reasons and despite apparent simplicity or homogeneity of Antarctic terrestrial and marine environments. Species defined by morphological characters are complexes of cryptic or sibling species that have been revealed by enormous genetic studies.

15.3 Genomics vis-a-vis Indian policy and regulations: current deliberations In India, the Department of Biotechnology, Ministry of Science and Technology, GOI, through their Review Committee for Genetic manipulation (RCGM) updated “Recombinant DNA safety guidelines, 1990” and “Revised guidelines for research in transgenic plants, 1998.” The RCGM has prepared detailed regulations and guidelines, i.e., “Regulations and Guidelines for Recombinant DNA Research and Biocontainment.” The guidelines are based on current scientific information as well as the experience gained while implementing the biosafety frameworks within and outside the country. The guidelines include import and export, storage, large scale manufacturing, facility certification, contained use (laboratory) and disposal and emergency related to recombinant DNA technology and use of hazardous organisms. The guideline document specifies the practices for handling hazardous biological material, recombinant nucleic acid molecules and cells, organisms, and viruses containing such molecules in order to ensure an optimal protection of public and occupational health and the environment. The document also provides clarity on biosafety requirements and recommendations for laboratory facilities (Bio Safety Laboratories: BSLs), including facility design, biosafety equipment, personal protective equipment, laboratory practices and techniques, waste management, etc. The regulations applicable to implement the provisions of Rules (1989) of EPA, 1986 for the manufacture, use, import, export, and storage of hazardous microorganisms, genetically engineered organisms or cells, which is applicable to the whole of India in the following specific cases: (a) To sale, to offers for sale, to store for the purpose of sale, to offers and any kind of handling over with or without a consideration; (b) Exportation and importation of genetically engineered cells or organisms (refer List of Risk Group microorganism in this document) (c) For production, for manufacturing, for processing, for storage, to import, drawing off, for packaging and repacking of the Genetically Engineered Products; (d) Production, manufacture, etc. of drugs and pharmaceuticals, and foodstuffs, distilleries and tanneries, etc., which make use of genetically engineered (GE) microorganisms one way or the other. The rules of 1989 are broad in scope. It covers the area of research, as well as large scale handling of hazardous microorganisms, GE organisms, and cells, and products thereof. In order to implement the rules in the entire country, six competent authorities and roles have been notified for: (i) Regulation and control of contained research activities with hazardous microorganisms, organisms, and cells, both GE and non-GE. (ii) Regulation and control of large-scale use of GE organisms in production activity. (iii) Import, export, and transfer of GE organisms and products thereof. (iv) Release of GE organisms in environmental applications under statutory provisions.

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15.3.1 Scope of guidelines The guidelines have the following scope: 1. Research: Any research toward the development of product or process involving Genetically Engineered (GE) organisms or non-GE hazardous organisms. 2. Large scale operations: Any scale-up processes toward large scale development. For large scale development at pilot scale and industrial scale, scheduled criteria like good large-scale practices (GLSP) are deployed for using recombinant organisms as per guidelines. It includes specific measures such as appropriate engineering toward containment, personnel protection, medical surveillance, and quality control, etc. 3. Environmental release vis-à-vis environmental risks: It includes field testing, the release of genetically engineered organisms into the environment. All regulatory measures are implied appropriately during release so as to ensure safety against environmental risks and hazards due to the release of genetically engineered materials, plants, and animals.

15.3.2 Classification of pathogenic microorganisms The classification of pathogenic microorganisms is defined under four risk groups, i.e., Risk groups 1e4 (RG1-4). The different risk groups are dependent on parameters like pathogenicity of the organisms, transmission mode, host range, available preventive and curative therapy, ability of making human/animals/plant diseases and strains causing an epidemic in India, etc. The parameters are controlled by immunity, density, and movement of the host population, vectors transmission, etc. During the handling of pathogenic organisms, the assessment of potential risks is done based on donor and recipient characterization, modified organism characterization and properties, and expression of the gene product. Different risk groups are related to different category (I, II, and III) experiments. Category I refers to experiments, where no significant risk group organisms are involved, whereas Category II refers to the involvement of low-risk group organisms and category III refers to the involvement of high-risk group organisms.

15.3.3 Containment The term “Containment” describes safe methods for managing infectious agents in the laboratory environment, wherein they can be controlled, handled, or maintained properly. The containment facility is recommended as per the World Health Organization (WHO) for Risk Group 1e4 organisms. The objective of containment is to reduce the risk and hazards of infectious agents to laboratory scientists, research workers, who are in exposure of RS 1e4 organisms. There are two types of containment viz. Biological containment (BC) and Physical Containment (PC). The vectors, including plasmid, organelle, virus for recombinant DNA (rDNA) transformation, and the host cell viz. bacteria, plant, and animal cell in which the vector is propagated in the laboratory are considered as biological containment. On the contrary, the physical containment is referred to physical parameters of the Containment facility protecting laboratory researchers and workers from exposure to infectious agents. It includes primary barriers like containment equipment and safety equipment, primary barriers like facility design, special laboratory design, laboratory practice, protection of personnel, immediate laboratory environment, and all biosafety levels, i.e., biosafety levels 1, 2, 3, 4, etc.

15.4 Mechanism of implementation of biosafety guidelines in India An institutional mechanism is very much needed for implementation of the guidelines and to ensure the compliance of requisite safeguards at various levels. The guidelines refer to establishing safety procedures for rDNA research, production, and release to the environment and setting up containment conditions for certain experiments. The institutional mechanism, as proposed for implementation of guidelines, consists of the following: (i) (ii) (iii) (iv) (v) (vi)

Recombinant DNA Advisory Committee (RDAC) Institutional Biosafety Committee (IBSC) Review Committee on Genetic Manipulation (RCGM) Genetic Engineering Approval Committee (GEAC) State Biotechnology Coordination Committee (SBCC) District Level Committee (DLC).

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While the RDAC has advisory in function, IBSC, RCGM, and GEAC are involved in regulatory functions. At the state/ district level, SBCC and DLC are responsible for monitoring the activities related to GMOs. RDAC, RCGM, and GEAC are constituted at the central level by DBT of Ministry of Science and Technology (MoST) and Ministry of Environment and Forest (MoEF). IBSCs are constituted at all organizations working in the area of GMOs, SBCC in all states, and DLCs in districts, wherever necessary. All IBSCs are empowered to review the applications related to research projects and submit their recommendations and reports to RCGM. After a comprehensive review, RCGM recommends to GEAC for scale-up activities, field trials, and environmental release. DLCs are also supposed to submit its report regularly to the SBCC/GEAC. In addition to the above committees, RCGM and GEAC constitute various subcommittees and expert committees’ case to case basis. Such committees are constituted by experts from various disciplines from public sector institutions to formulate and review various guidelines and biosafety parameters. Central Compliance Committees are also developed for review and monitoring of confined field trials on case to case basis.

15.4.1 Recombinant DNA Advisory Committee (RDAC) This committee involves the national and international levels biotechnological developments toward the completeness and correctness of the safety regulation for India on recombinant research (rDNA), its uses and applications. The TOR (terms of reference for Recombinant Advisory Committee) is as follows: (i) Evolving long term policy on R&D (research and development) in r DNA research. (ii) Formulation of the safety guidelines for rDNA Research to be followed in India. (iii) Recommendation of type of training program for Research scientists, technicians, research assistants, and research scholars to adequately become aware of hazards and risks involved in rDNA research and the process of avoiding that.

15.4.2 Institutional Biosafety Committee (IBSC) Institutional Biosafety Committee (IBSC) should be constituted in all centers engaged in genetic engineering research and production activities. The Institutional Biosafety Committee (IBSC) is mandated to implement the guidelines within the institution. Any research project having biohazard potential or production of either microorganisms or biologically active molecules causing biohazard should be informed to IBSC. IBSC will review the project and may approve the execution of the project at notified places as per guidelines. The approval includes the safe, storage facility of donor, vectors, recipients, other materials, and processes used in the experimental work subject to requisite inspection and accountability as per guidelines.

15.4.3 Review Committee on Genetic Manipulation (RCGM) The RCGM has the following mandates: (i) To develop a procedural manual, including the regulatory process toward research, production, and its application for genetically engineered organisms in relation to environmental safety. (ii) To review the IBSC approved ongoing project reports involving controlled and high-risk category field experiments so as to maintain the safeguards as per guidelines. (iii) To recommend types of containment facility and also the special containment conditions to be followed as per experimental trials and also for certain specific experiments. (iv) To advise customs Authorities of Central Revenue and Excise department regarding guidelines and procedure on import of biologically active material, genetically engineered substances or products, including details about excisable items. (v) To suggest Banks, Ministry of Commerce and Industry, and other relevant agencies regarding clearance of documents toward setting up industries on genetically engineered organisms. (vi) To help and support the BIS (Bureau of Indian Standards) in evolving standards of biologics developed through rDNA technology. (vii) To advise intellectual property rights with respect to rDNA technology on patents.

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15.4.4 Genetic Engineering Approval Committee (GEAC) Genetic Engineering Approval Committee (GEAC) will function under the Department of Environment (DOEnv) as a statutory body for review and approval of activities involving genetically engineered organisms and their products for scale-up R&D, industrial production, environmental release and field applications. The functions of GEAC include approval from the environmental angle on the following points: (i) Importing, exporting, transporting, manufacturing, processing, and selling of any products, including foodstuffs and additives that have been developed through Genetically Engineered (GE) microorganisms, substances, or cells using gene therapy technology. (ii) Releasing GE classified cells or organisms into the environment from the research laboratory, hospitals, field, and related areas. (iii) Production of large-scale GE microorganisms, substances, cells in industry, and its related applications. (iv) As a special case, the deliberate release of GE organisms with an approval period of 4 years. GEAC consists of Biotechnology Coordination Committees, which will function as a legal and statutory body with judicial powers to inspect, investigate and take punitive action in case of violations of statutory provisions under the Environmental Protection Agency (EPA). The provisions are as follows: (i) During the handling of large-scale GE organisms for scale-up research, developmental, and industrial production, rigorous review and control of safety measures are to be adopted. (ii) During the large-scale release of GE organisms or GE products into the environment for experimental field trials or other field applications, rigorous monitoring is to be adopted. (iii) Information and data upon surveillance with respect to safety, risks, and accidents against approved projects for industrial production and environmental releases are to be provided to RCGM.

15.4.5 State Biotechnology Coordination Committee (SBCC) SBCC is constituted in each State where research, development, applications, the release of GMOs are underway. SBCC is primarily mandated for monitoring responsibilities and is headed by the Chief Secretary of the State. The SBCC has the following powers: I. Inspection, investigation, and taking punitive action against violations of statutory provisions through Directorate of Health and State Pollution Control Board (SPCB). II. To review periodically the safety and control measures established at various institutions handling GE organisms. III. To act as a nodal agency at the State level to assess the damage, if any, due to the release of GE organisms and to take on-site control measures.

15.4.6 District Level Committee (DLC) DLCs are constituted at the district level to monitor the implementation of safety regulations in relation to the use of GMOs/hazardous microorganisms, and its applications in the environment. Each DLC is headed by the officer responsible for the administration of a district, i.e., District Collector. In addition, the officers concerned with public health, environment, pollution control, etc. at the district level are also included in the DLC. In Rules (1989), the mechanisms of interaction between committees have been provided. The DLC has the following powers: I. The District Level Committee/or any other person/s authorized in this behalf shall visit the installation engaged in an activity involving genetically engineered organisms, hazardous microorganisms, formulate information chart, find out any risks and hazards in relation to each installation followed by taking the necessary remedy as an emergency. II. They shall also prepare an off-site emergency plan. III. The District Level Committee is supposed to submit the regular report to the SBCC and GEAC

15.5 Assessment of environmental risk It is acknowledged that Genetically Modified (GM) animals are more likely to pose a risk of damage to the environment than they are to human health. The Environmental Protection Act defines damage very broadly as being caused by the presence of Genetically Modified Organisms (GMOs) that have escaped from containment and are capable of causing harm

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to any living organisms supported by the environment. Therefore, the primary objective of the risk assessment is to determine the likelihood and the possible consequences of an accidental release of a GM animal from containment into the environment. With the correct containment measures in place and an appropriately maintained and managed facility, the risk and hazards to the likelihood of such a release will be low. However, identification of all possible hazards and consideration of any routes by which a GM animal could escape is important to follow. The concern is for GM animals that could feasibly cause harm to the environment, particularly those that could impact on any environmental ecosystem (including indigenous plant and animal populations). With the modification of the intrinsic characteristics of the animal species, the risk assessment may be increased or decreased. It may be correlated with genetic modification itself. However, in some cases, the hazards are coming due to genetic modification directly and also interact directly with the characteristics of recipient species.

15.5.1 Mechanisms by which the environment may be exposed to GMO hazards In the hazard identification process, the factors which are to be considered are as follows: l

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The ability of the GM organisms or GM animals to become, survive, establish, and disseminate. This includes its capacity to compete with other animals or discharging other adverse effects on animals and other populations. Risks and hazards associated with the inserted gene or gene fragment, which encodes a toxic product, and therefore, may pose an adverse effect due to its biological functions Possibility of transferring genetic material between GM animal and other organisms Stability in respect of phenotypes and genotypes.

The above factors may have the possibility of posing risks or hazards in respect of GM animals, including recipient organism, the insert, and the final GMO. It is necessary to evaluate the transmission of genes from one organism to another within sexually compatible species. It is also necessary to evaluate the potential consequences of transmission of genetically modified organisms, including its synergistic and cumulative effects.

15.6 Capacity to survive, establish and disseminate l

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The survival capability of a GM animal is one of the most important and key attributes toward identifying potential risk factors affecting the environment. It is noted that GM animal may not cause environmental harm if the animal is unable to survive outside of containment. For example, Tilapia is a freshwater fish native to Africa and unlikely to survive in UK waters, as it requires ambient temperatures of above 27
C. It is, therefore, effectively under innate biological control. Due to the presence of GM animals in the environment may cause harm. If they are adapted to the climate and environment, they may compete and displace other populations of animals or prey upon native populations. The important point affecting survivability and dissemination in the environment is the ease with which there is a possibility of recovering escaped animals. For large animals such as sheep or pigs, it should be feasible to retrieve escapees. However, small organisms like insects, fish, and small mammals, it is very difficult or even impossible to recover. In particular, the small size and short reproductive cycles of many of these animals may mean that the GMO could become disseminated very rapidly. The complex life cycle seen in many invertebrates includes additional pathways for escapisms and dissemination, wherein it is very difficult to track and recover them.

15.7 Hazards associated with the inserted gene/element l

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It is likely that modified animals for producing biologically active substances are used for the expression of molecules for pharmacological use like tissue plasminogen, blood clotting factors or insulin-like hormones, etc. It is also likely that the inserted gene in modified animals could possibly be harmful to other animals. In fact, there is a possibility of genetically modified animals expressing biologically active substances that may be allergenic, immunogenic, or toxic. Therefore, it will be exposed to whole natural habitats, including affecting animal populations too. It is necessary to give appropriate attention to the GM animals that may act as vectors or reservoirs of novel animal diseases. For example, if an animal is modified to express a receptor for a particular virus, those animals may be able to act as a reservoir for that. Similarly, it should be considered that species modified to become susceptible to specific pathogens could disseminate that disease.

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15.8 Transfer of harmful sequences between organisms l

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It may be taken into consideration that the possibility of transferring inserted genetic material to other organisms, which could be present in the receiving environment. It is likely to be dependent on sexually compatible species. If GM animal is unfertile, then there is no possibility of transmitting inserted genes through the germline, unless there is the restoration of fertility due to some unique genetic reversion. The nature of the risk and hazards obtained from genetic modification and subsequent transfer may also be considered appropriately. If it is observed that the modification is posing negative effects on animals in respect of their health or longevity, there will have enormous deleterious selection pressure.

15.9 Phenotypic and genetic stability l

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Genetic instability results in loss of inserted gene or any other associated traits, which may rarely be a source of potential harm. But genetic stability is essentially considered if the purpose is for large biological containment toward the development of GM animals, and eventually, those restricted characteristics could revert to wild type. In that context, the genetic stability of such modification may be inextricably linked to phenotypic stability, wherein it restricts the GM animal’s ability to survive and to spread. If an organism is surviving with the restricted capacity, it is expected that the organisms will be under stress in the environment. Subsequently, a strong selection pressure will be implied in favor of reversion. The possibility that the genotype of a modified animal will be unstable in the environment should be taken into account and consideration given to any detrimental effects this might cause.

15.10 Risk assessment for human health The Contained Use Regulations is a requirement to consider risks and hazards toward human health created by the GM activity. The objective is to identify all possible hazards to human health, and assess the likelihood and potential severity of the consequences. It is recognized that many activities with GM animals pose no additional risk to workers over those that would be expected from the unmodified animal. Some hazards are intrinsic in nature, which includes allergenicity, bites, scratches, and zoonotic, which are common to all animals. The GM risk assessment should identify any additional hazards against human health, which may come directly due to genetic modification. Due to modifications, human hazards might arise, which mainly affect allergenicity or toxicity.

15.11 Mechanisms by which the GMO could be a risk to human health l

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As far as environmental risk assessment, the hazard identification process must include considerations of potentially harmful or adverse effects to human health that would be mediated by the final GMOs, which is actually a product developed through inserted genes. As an example, if an animal line has been produced through modification like containing a receptor for a human virus, those animals may act as a novel reservoir for human disease. Although there is a possibility of such additional human hazards as above, in most cases, such genetic activities will not necessarily pose any extra hazards to humans. Additional risks to humans will most likely arise due to modifications that alter allergenic or toxic properties. Such hazards might include: l novel or increased allergenicity; l possible toxic effects due to the production of toxins or other biologically active proteins; l adverse effects due to altered behavior (like enhanced aggression, etc.).

15.12 Control measures to protect human health l

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It is necessary to evaluate whether any specific control measures are required to adequately protect human health or not. If necessary, containment measures should be applied until the risk of harm become “effectively zero.” It is a requirement of the Contained Use Regulations that all measures deemed by the risk assessment as necessary for the protection of human health need to be implemented. Following principles of good occupational safety and hygiene will be sufficient to protect human health in major cases.

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After several deliberations, consultations of national and international experts and stakeholders, RCGM of DBT, MoST, India, has published comprehensive guidelines on “Regulations and Guidelines for Recombinant DNA Research and Biocontainment, 2017.” The Guidelines cover the regulations on biosafety of recombinant DNA (rDNA) research, development, working in laboratories and animal facilities, trial, validation, release, and handling of hazardous microorganisms and GMOs in India. It includes containments, including details of principle, factors, and scope, etc. The containments include Physical Containment, Procedure, Safety Equipment Facility Design, Biological Containment, Laboratory Monitoring, Health and Medical Surveillance, Decontamination and Disposal and Emergency Procedures. It also provides clear deliberations on disposal and decontamination of laboratory wastes, emergency procedures, etc. The guidelines provide a spectrum of handling of microorganisms, animals, plants, insects, and aquatic organisms. The guidelines include lists of risk group (1,2,3 and 4) agents vis-à-vis appropriate containment level for implementation or handling in India. As per rules, IBSC (Institutional Biosafety Committees) is empowered to take adequate precautionary measures for research conducted on risk groups 1 and 2 organisms. However, RCGM approval is required for experiments involving risk groups 3 and 4 organisms. In addition, for handling the organisms of risk groups 3 and 4, a separate laboratory certification system has been developed. For the implementation of guidelines for working with risk groups 3 and 4 organisms, facilities like biosafety levels 3 and 4 are mandatory along with existing high containment facilities to acquire relevant accreditation. Adoption of these guidelines shall be binding pan India for all public and private organizations involved in research, development, and handling of GE organisms (organism includes microorganisms, insects, arthropods, animals, plants, and aquatic animals, etc.) and non-Genetically Engineered (GE) hazardous microorganisms (viz. prions, virus, bacteria, parasites, protozoa, algae, and fungi, etc.) and products developed through exploration of such GE or non GE hazardous organisms. The principles related to rDNA research are detailed in “Regulations and Guidelines for Recombinant DNA Research and Biocontainment 2017.” In addition, some of the measures that need to be implemented for environmental protection may be adequate to minimize or prevent exposure. However, only risks to human health will have a bearing on the notification requirements for the work vis-a-vis workplace or facilities. In India, “Regulations and Guidelines for Recombinant DNA Research and Biocontainment, 2017” issued by Department of Biotechnology, Ministry of Science and Technology, Govt. of India, also refers comprehensive operational guidelines, including biosafety levels for microbiology (general/hazardous microorganisms), GE microorganisms, animals, plants, insects, and aquatic organisms.

15.12.1 Biosafety Level (BSL) facilities The facility includes Biosafety Level 1 (BSL-1) for experiments on Risk Group (RG) one microorganisms not derived from pathogens, Biosafety Level 2 (BSL-2) for experiments on Risk Group (RG) two microorganisms collected from an environment that is unlikely to contain pathogens, Biosafety Level 3 (BSL-3) for experiments on Risk Group (RG) three microorganisms samples collected from an environment that is likely to contain pathogens of potential disease consequences and Biosafety Level 4 (BSL-4) for experiments on Risk Group (RG) four microorganisms collected from environment/patients that are likely infected organisms with serious/fatal health effects. This kind of facilities will be suitable for: l l l l

To isolate, cultivate, storage of Risk group 1 to Risk group 4 (RG1-RG4) microorganisms Genetic engineering of organisms (RG1-RG4) and their safe handling. Safe handling of toxins, tissues, etc. Experiments of micro from Risk group 1 to Risk group 4 organisms, including hazardous on RG4 microorganisms

The operational guideline includes facility design (controlled access through the controlled air system, cabinet room, suit laboratory, etc. for BSL-4), safety equipment, Personal Protective Equipment, Procedures, Laboratory monitoring, Waste management, Health and Medical Surveillance, and Emergency procedures, etc.

15.12.2 Different Bio Safety Level nomenclatures The comprehensive operational guidelines include biosafety levels for microbiology (general/hazardous microorganisms), GE microorganisms, animals, plants, insects and aquatic organisms. However, the nomenclatures are different from group to group. For general, hazardous and GE microorganisms the nomenclatures are BSL (1e4), wherein for animals it is known as Animal Bio Safety Level 1e4 (ABSL1-4), for the plant it is known as Plant Bio Safety Level 1e4 (PBSL1-4), for insects it is known as Insects Bio Safety Level 1e4 (IBSL1-4) and for Aquatic Organism it is known as Aquatic

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Organism Bio Safety Level 1e3 (AqBSL1-3). It is claimed to have no pathogens for BSL-4 type containment for aquatic organisms, i.e., AqBSL-4, however, sometimes, to designate a pathogen as requiring AqBSL-4 level is done on a case to case basis. The Animal Bio Safety Level 1e4 (ABSL1-4) are detailed as below:

15.13 Animal Bio Safety Level (ABSL) facilities The facility is implemented in animal houses developed research purposes. Those animals are used for testing of chemical drugs or risk-inherent microorganisms or sell as laboratory animals. The ABSL facility ensure that l l l

Healthy animals are not getting an infection, followed by clinical disease or mortality. Within the same facility, it protects the spreading of infections to other animal houses. To prevent zoonosis from animals to laboratory workers through bites, scratches, and inhalations of aerosols.

For experimental, research, and development purposes, working with animals should first satisfy legislative obligations set in the Prevention of Cruelty to Animals Act, 1960, Breeding and Experiments on Animals (Control and Supervision) Rules of 1998, 2001, 2006. The constitution of the Institutional Animal Ethical Committee (IAEC) and their approval toward any research projects with animal experiments are mandatory. As per obligation, in each step of the experiment, taking care of an animal, humane approach, including avoiding unnecessary pain or suffering, is expected. Animals must be provided a relaxed, hygienic, and comfortable stay and adequate food and water. It is noted that ABSL facilities is only used for (a) animals that are used in the intended experiments. (b) neither of plants, arthropods, nor aquatic organism’s housing/keeping/rearing unless they are an integrated activity of the designed animal research in the contained facility.

15.13.1 Operational guide for ABSL facilities Operational guide for ABSL facilities includes detailed modalities, including parameters like facility design, safety equipment, Personal Protective Equipment, Procedures, Laboratory monitoring, Waste management, Health and medical surveillance, Emergency procedures, etc. The modalities under each parameter are almost similar to BSL modalities applied for microbiology (general/hazardous microorganisms), GE microorganisms. However, the details regarding the operational guide on containment, which is followed in India, are captured in Chapter 3 of “Regulations and Guidelines for Recombinant DNA Research and Biocontainment, 2017” issued by Department of Biotechnology, Ministry of Science and Technology, Government of India.

15.13.2 Types of Animal Biosafety Level facilities 15.13.2.1 Animal Bio Safety Level 1 (ABSL-1) It is deployed for purposes as follows: l

l l

For maintenance of post quarantine stock animals (except nonhuman primates, for which the appropriate National authorities should be consulted). For breeding, housing, and experiments with animals that are deliberately inoculated with RG one microorganism. For genetic engineering experiments on animals as Category I experiments. The category includes experiments that do not show significant risks for research workers, including laboratory workers, the community or the environment. The examples are included as (i) Breeding of GE animals with viral vector sequences transformation belonging to RG 1. (ii) Experiments of gene “knockout” in rodents, its further breeding, housing, management. It is independent of carrying a selectable marker gene by the mice, provided that the marker gene does not indicate any selective advantage to the animal. However, containment might be decided after following a thorough risk assessment, if further genetic manipulations are performed on those “knockout” mice. (iii) Research and development, including the insertion of nucleic acids into animals, provided that the nucleic acid does not give rise to any infectious agent. (iv) Research, including the introduction of somatic cells with genetic manipulation into animals, provided that they are unable to give rise to infectious agents.

It is mandatory that the investigator must intimate IBSC regarding his project, including the objective and experimental design of the study and the organisms involved before the commencement of the Category I GE experiments. IBSC has been mandated to review the project and recommend action, if any. For Category I GE experiments, it is advisable for a dedicated area in the facility with proper labeling to avoid any chances of contamination.

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15.13.2.2 Animal Bio Safety Level 2 (ABSL-2) The purposes of ABSL-2 are as follows: l l

The experiments with animals (including breeding and housing) that are inoculated with RG two microorganisms. For genetic engineering experiments on animals as Category II experiments. The category II experiments refer to lowlevel risks to laboratory researchers, including laboratory workers, the community, or the environment. The examples are included as (i) R&D experiments with GE animal and related materials, wherein DNA from RG two microorganism has been inserted. (ii) R&D experiments with vertebrate and nonvertebrate animals, involving stable genetic manipulation of oocytes, zygotes, or early embryos to produce a novel organism. (iii) R&D experiments with animals infected with RG two genetically engineered microorganism(s).

It is also mandatory that before the commencement of the experiments, all Category II GE experiments require prior approval from IBSC in the prescribed proforma. For Category II GE experiments, it is also advisable for a dedicated area in the ABSL-2 facility with proper labeling to avoid any chances of contamination.

15.13.2.3 Animal Bi Safety Level 3 (ABSL-3) The purposes of ABSL-2 are as follows: l l

For experiments with animals (including breeding and housing) that are inoculated with RG two microorganisms. Genetic engineering experiments on an animal that is Category III and above. The category III experiments pose moderate to high risks to laboratory scientists, research workers, including the community or the environment. The examples under this category are: (i) Breeding, housing, management, and R&D experiments with animals infected with RG three genetically engineered microorganisms. (ii) R&D experiments involving the use of infectious or defective RG three viruses in the presence of helper virus. (iii) Experiments on animals using DNA that encodes a vertebrate toxin. (iv) Experiments using viral vectors with inserted DNA sequences coding for a known product where the host range includes humans. The effect is to play a role in the regulation of cell growth or toxic to human cells. (v) Any R&D experiments using defective vector and helper virus combinations having the potential to regenerate nondefective recombinant virus. (vi) Introduction of genes producing pathogenicity into microorganisms other than the host organisms. Such genes are suspected of developing products having a risk of autoimmune diseases. (vii) It also includes cloning or transfer of fragments or entire viral genome capable of giving rise to infectious agents infecting humans, animals, or plants. (viii) R&D experiments involving recombination between complementary fragments or entire viral genomes, where one or more fragments encode virulence or pathogenic determinants. The experiments that may alter the host range of pathogens or increase pathogen virulence or infectivity are as follows: (ix) R&D experiments where the entire genome or fragment of a virus is injected into an embryo to produce a transgenic animal producing infectious viral particles. (x) Experiments with animals infected with GE microorganism(s) that fall under RG 3.

It is mandatory that all category III GE experiments need prior approval from IBSC and RCGM before the commencement of the experiments through prescribed proforma.

15.13.2.4 Animal Bio Safety Level 4 (ABSL-4) It is implemented for the experiments as below: (i) Breeding, housing, management, and R&D experiments of the animals that are experimentally inoculated with RG four microorganisms. (ii) The animal experiments with genetic engineering on animals involving GE microorganisms of above Category III, which fall under RG 4. It is mandatory that all GE experiments above category III, need prior approval from IBSC and RCGM before the commencement of the experiments through prescribed proforma.

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15.14 Approvals and prohibitions The Rules 1989 capture all possible compliance related to biosafety safeguards. Any violation or noncompliance attracts punishment under EPA 1986. As per provisions of Rules (1989), the approvals and prohibitions are appended below: I. Without the approval of GEAC, no person is allowed to import, export, transport, manufacture, process, use, or sell any GMOs, substances, or cells. II. As per provisions of EPA, 1986, pathogenic organisms or GMOs or cells are allowed to use for research purposes only in laboratories or inside laboratory areas notified for this purpose as per rule. III. Any person operating or using GMOs for scale-up or pilot operations shall have to obtain permission from GEAC. IV. Experiments for the purpose of education involving GMOs can be undertaken with the oversight of IBSCs. V. Deliberate or unintentional release of GMOs not allowed. VI. Production in which GMOs are generated or used shall not be commenced except with the approval of GEACA VII. All approvals shall be for a period of 4 years, at first instance, renewable for 2 years at a time. VIII. GEAC shall have powers to revoke approvals in case of: i. There is any such new information through GMOs, or any harmful effects are observed. ii. Approved GMOs are causing damage to the environment as it could not be envisaged when the approvals toward those GMOs were given. iii. Noncompliance of any conditions stipulated by GEAC.

15.15 Conclusion In the modern era, with the advancements of science and technology, there are enormous variations in animal genomics issues. Regulatory policies are, in general, compliance-friendly. Biotechnological applications for livestock should be suitable for animal owners having poor, small-scale operators having little or no land but have few animals in developing countries. In developing countries, livestock production is increasing rapidly as a result of growth in population and incomes and changes in lifestyles and dietary habits. In line with growing food demand, which is expected to be increased by up to 40% by 2030 and around double by 2050, the role of the livestock sector is very much important. The wide gap between demand and availability and scarcity of resources are putting more challenges for increasing the productivity and/ or reduce the losses. The effective production and productivity of agriculture, including animal husbandry, is essentially required to be improved. It will substantiate the ever-increasing consumer demand and generate sufficient income for the emerging agricultural population. The last three decades have witnessed tremendous growth in technological advancements in different disciplines of Science and Technology. Modern biotechnology is a panacea for all the problems that farmers face. Genetic engineering has the potential to improve the speed of development. It also provides compelling benefits to transform public health, including improved foods, advances for human health, enhanced animal welfare, and reduced environmental impact. The development of technology for sequencing of the whole genome and mass screening of gene regulation has widened our approach to the genetic profiling, mapping, as well as furthering our understanding of underlying biological mechanisms. Advances in animal genomics through transcriptomics, proteomics, metabolomics with the help of robust genetic engineering tools animal production through GMOs will definitely bring out exciting changes. Faster rates with higher yield in all aspects of animal production, including tailoring of animals, fulfill great human demand and will try to make shorter the demandesupply gap. It is well known that the creation of a new genetically engineered animals line needs invasive (surgical) procedures like the intraperitoneal or subcutaneous injection of hormones for multiple ovulation, vasectomy, surgical embryo transfer of genetically engineered embryos at recipients, offspring genotyping taking tissue samples or tail biopsies, including the sacrifice of animals. Those procedures may not be unique to genetically engineered (GE) animals, but they are typically needed for GE animal production. It is also noted that a large percentage of genetically engineered embryos do not survive, only a small proportion (between 1% and 30%) carry the genetic alteration of interest. Therefore, the ethical issues related to invasiveness of procedure, as well as the requirement of a large number of animals, are an important unanticipated animal welfare concern, Ethical issues, including animal welfare concerns can arise at all stages in the generation and life span of an individual genetically engineered animal. There are other ethical issues in the fields of Genetic engineering like intellectual property, patenting of created animals and/or the techniques used to create them, application of genetically engineered animals, etc. There are restrictions on the methods of their disposal also once they have been euthanized. The reason for the restriction is to restrict the entry of genetically engineered animal carcasses into the natural ecosystem since its long-term effects and risks are not better understood. The concerning issues related to guidelines in relation to

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restrictions on the methods of their GE animal research, production, release, commercial marketing, and disposal are addressed, although several National and Global policies have been discussed above. GE engineered animals have brought extra challenges in respect of animal welfare impact in the population and also in the environment. Several governing bodies increased vigilance and monitoring to understand potential animal welfare impacts through the implementation of relevant policies. It has got an impact to inform the public about genetic engineering techniques and any potential impacts on animal welfare and food safety today or in the future. In this context, the implication for veterinarians is gradually increasing since genetically engineered animals are entering into the commercial realm. It will become increasingly important for veterinarians to inform themselves about any special care and management required by those animals. It is assumed that animal health professionals, veterinarians can contribute a lot in policy discussions related to the oversight of animal genetic engineering and also in various regulatory proceedings toward commercial use of genetically engineered animals, especially those animals used for food production. Genetic engineering techniques are applied to a range of animal species. Many are in the research phase, although there are a variety of intended applications for their use. It is true that in biomedical science and food production, genetic engineering may provide substantial benefits. But the creation and use of genetically engineered animals not only offer challenges but may also raise several ethical issues related to animal integrity and/or dignity that go beyond considerations of animal health, animal welfare, etc. Therefore, there are robust policies (as above) related to animal welfare are implied by concerned Government departments, which can be safeguarded satisfactorily on intrinsic ethical concerns about the genetic engineering of animals. The policies are planned to restrict certain types of undesirable genetically engineered animals from reaching their intended commercial application. India is a signatory to the Cartagena Protocol on Biosafety (CPB); however, the inclusion of modern biotechnology, as per modern definition is needed to be revamped in the national regulations appropriately. In India, the policies are strictly adhered to by concerned government bodies like the Ministry of Science and Technology, Ministry of Environment and Forest, Ministry of Health and Family Welfare, and different National, State, and District level Committees toward the implementation of animal genomics and its several issues. With the above regulatory mechanism so far around 10 r-DNA drugs have been approved for marketing; around four industrial units are manufacturing recombinant hepatitis vaccines, erythropoietin, and G-csf, locally and indigenously, and some of them are in the market. It is known that there are many novel processes toward the development of r-DNA vaccines and drugs in advanced stages. In the case of plants, cotton with insect-resistant Bt gene has been given approval for licenses, colicenses for commercial release from March 2002. More than 500 institutions (public and private) are working in r-DNA research by constituting Institutional Biosafety Committees (IBSC). As per the survey of MoEFCC during 2014, over 85 different plant species were identified as being used in GM experimental work, including plants used for food, livestock feed, fiber fuel, and dietary or medicinal purposes. For GM food, the detection of genetic modification and analytical food safety laboratories have been established to facilitate the generation of scientific data, appropriate validation for human and animal use. Similarly, containment facilities at biosafety levels 1, 2, 3, and 4 are also available for both for research development and evaluations. In India, stepwise regulatory procedures/protocols have five categories I. Protocol-I: Indigenous product development, manufacture, and marketing of pharmaceutical products derived from LMOs (Live Modified Organisms), but the end product is not an LMO. II. Protocol-II: Indigenous product development, manufacture, and marketing pharmaceutical products where the end product is a Live Modified Organism (LMO). III. Protocol-III: Import and marketing of LMOs as Drugs/Pharmaceuticals in finished formulations where the end product is an LMO. IV. Protocol-IV: Import and marketing of LMOs as Drugs/Pharmaceuticals in bulk for making finished formulation where the end product is an LMO. V. Protocol-V: Import and marketing of the products like Drugs/Pharmaceuticals bought in bulk and/or finished formulations, which have been derived from LMOs, but the end product is not an LMO. Expected Timelines for approvals in India: I. II. III. IV.

RCGM approval for preclinical animal studies: Around 45 days RDAC approval for Human Clinical Trials protocol: Around 45 days RDAC (DCGI) examination of trial data and approval: Case-specific Simultaneous DCGI and GEAC* approvals: Around 45 days

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V. GEAC approval procedure will be compliant with the “Good Practices in Environmental Regulation” adopted by MoEF. However, in the process flow, it is proclaimed that too many agencies are involved in regulatory clearances. For overcoming the issues raised by both public and private sectors, necessary policies are under process to establish a singlewindow regulatory mechanism and/or toward making a structure to promote speedy commercialization of recombinant products and processes. For establishing a single-window system, many countries reviewed the merits and drawbacks of regulations in terms of complexity, timeliness, flexibility, and cost. However, realizing the spectrum of GMOs at present and predicted for the future, it would be relevant to have a separate regulatory authority for biosafety issues of GMOs. The structure, functions, and operational cost of such regulatory authority through a new legislation may differ country-wise based on the technoeconomic situation and the needs in terms of research, trade, and industrial activity. If any country follows a single-window regulatory authority and policy, it should cover recombinant products of agriculture, healthcare, veterinary, and others. It should also cover all five types or protocols as above (IeV), safety issues at all levels, including research, development, production, release, marketing, import, export, interministerial coordination, etc. Therefore, it has been identified that if there is perfect coordination and timeliness among different ministries involved, probably no new legislation is required since it is difficult to amend new legislation compared to implementation procedures of biosafety policy within the existing law. Constitution of Interministerial committees may be a good practice for addressing the issues with speed and success if interministerial coordination is very weak and time-consuming. As of now, the overall system is relatively open and transparent with the requisite precautionary approach. There is enough expertise in technology and risk assessment of GMOs and therapeutics in terms of safety to the environment, as well as human and animal health. In line with recent trends and public perception, appropriate measures and mechanisms are being evolved to label the GMOs, including GM foods and therapeutics, within the scope of CODEX Alimentarius. However, time-to time revisions and modifications of the Acts, guidelines, and procedures based on stakeholder’s feedback and science-based developments in risk assessment are quite common practices for all Nations. The intension is to create a more streamlined and certain pathway for industry and researchers seeking approval of GMOs that can be managed safely. Biosafety policy and procedures affect diverse group of stakeholders. The decisions taken by the biosafety system are subjected to local, national and even international examinations. In addition, it also concerns researchers, developers, producers, consumers, environmental groups, NGOs, nontechnical groups and other ministries of the government. In view of the complexity of views regarding genetic engineering, it is valuable to involve all stakeholders in discussions about the applications of this technology. There have been many suggestions to address the involvement of all stakeholders in the biosafety decision-making process. Besides being transparent, the system should be amenable for addressing various crosscutting issues affecting specific stakeholder groups. Apart from stakeholders, the cost-effectiveness parameter in the development of GMOs is another important issue. Generally, in a country, there are two types of expenses involved in the establishment and maintenance of the biosafety system. The first one is incurred by a regulatory agency/department, and the second by the client interested in the commercialization of GMOs. The clients are requested to incur expenditure toward various tests related to toxicity, allergenicity, and small and large field trials, including R&D prototyping, safety assessment. The total amount is varied for different countries on a case-to-case basis. The harmonization of regulatory procedures around the world is very important. The regional harmonization in most of the cases is related to the basic procedures and common interest. It may facilitate the cost-effectiveness of regulatory requirements through regional cooperation. Regional cooperation in biosafety may be deployed through joint food and environmental safety experiments, compatible regulations to resolve cross-border problems, sharing expertise, information, facilities, training, and education, etc. International agencies like Food and Drug Administration (FDA), United Nation Environmental Program/Global Environment Fund (UNEP/GEF), Organization for Economic Cooperation and Development (OECD), European Union (EU), Food and Agricultural Organization (FAO) and International Service for National Agricultural Research (ISNAR) have been active in capacity building, harmonization of biosafety regulation in different regions. Those organizations have published Consensus documents on various specific issues of regional importance. With the advancement of drug and pharmaceutical research and development, safety guidelines toward assessment of toxicity, allergenicity, impurity are already in place from long ago. Safety issues related to food and environment have adequately been addressed by all over the world. Therefore, both developed and developing countries have considerable agelong experience in procedures, implementation, and enforcement of guidelines related to non-GMO products. However, the regulations of GMOs are of recent origin, and it needs adequate time for the stabilization of procedures, framework and guidelines. Continuous feedback from all stakeholders considering social, economic, other nonscientific factors, international developments, trade and commerce will help to revamp existing GMO safety guidelines as comprehensive and

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rational. Due to rapid shifts in industrialization, India clearly recognizes the need to restructure its GMO regulatory system so as to compete with other global players in international markets. Since India is a signatory to Cartagena Protocol on Biosafety, the modern biotechnology Protocol is needed to be adopted through the establishment of the Biotechnology Regulatory Authority of India. In the present definition, the regulatory considerations for new and emerging technologies are reviewed on a case-to-case basis and considered accordingly under the existing regulatory framework. While developing the national biosafety system, a country must decide the scope of the system with necessary deliberation to speed up the process, possibly single-window systems, etc. The three components that may be treated as major portion of the protocol like the need of transboundary movement of transgenic products, use of principles, which are precautionary in nature, and capacity building have several implications for individual countries as users, developers, and exporters. Understanding the need, during 2014, DBT constituted a dedicated Task Force on “Genome Engineering Technologies and their Applications.” The objective was to foster innovation and promote the development of Genome-wide Analysis and Engineering Technologies and to revamp the policy to make them accessible and affordable for wider use in life sciences. During revamping of safety guidelines, cooperation between agencies within the country and International or regional harmonization of regulations is quite important. Creation of capacity building in terms of infrastructure and expertise for biosafety experiments, resolving public concerns, maintaining databases, monitoring GMOs, their commercialization, performance, and safety assessment are important issues to be taken care of at the National policy level. Updating knowledge of main stakeholders involved in regulatory committees/ agencies is the immediate rush for evolving a revamped framework for biosafety implementation with wider public acceptance. Genome engineering technologies, especially issues and policies related to animal genomics, still confront debates in India. The Rules and Acts are very broad-based and include “modification, deletion or removal of parts of heritable material.” It is suggested to improve facilities on emerging latest technologies like rDNA, gene editing, and other similar types of research projects. Recognizing the global advances and understanding the huge potential for practical applications in healthcare, agriculture, and animal genomics, novel efforts should be implied to resolve emerging issues. Innovative incorporation at regulation and policy level with a single-window-based user-friendly mechanism is demanded in the present era to make faster growth. It may synergize the benefits of these technologies for basic or applied use for animal genomics in a larger context. It attracts faster growth in animal genomics, and it is expected to uplift the potential and prospects in the overall growth of global animal proteins.

Disclaimer The paper has been written in his personal capacity and the views do not necessarily represent those of DSIR, any public, private, society, trust or other organizations nationally/internationally.

Conflict of Interests The author declares that there is no conflict of interests regarding publication of this paper as a book chapter

Acknowledgments Writing a book chapter is tougher than we thought, but it is more rewarding than our imagination. As a first author, I can acknowledge, none of this would have been possible for me without getting opportunity of working on regulatory, innovation, technology development and societal implementation areas at Department of Scientific & Industrial Research (DSIR), Ministry of Science & Technology, Government of India. The authors also acknowledge contribution and initiatives of different scientists, officials of Ministry of Environment, Forest & Climate Change (MoEF&CC), Department of Biotechnology under Ministry of Science & Technology (DBT, MoST), Ministry of Health and Family Welfare (MoHFW), Ministry of Agriculture & Farmers Welfare (MoAFW), National Biodiversity Authority (NBA) and Food Safety and Standards Authority of India (FSSAI) etc. in formulating different GM rules, Acts, Guidelines which helpsto frame this chapter in a structured manner.

References DBT and MoEFCC, 2008a. Guidelines for Confined Field Trials of Regulated GE Plants, pp. 1e14. DBT and MoEFCC, 2008b. SOPs for Confined Field Trials of Regulated GE Plants, pp. 1e50. Department of Biotechnology, Ministry of Biotechnology. Govt. of India, 2008. Protocols for Food and Feed Safety Assessment of GE Crops, pp. 1e38. Indian Council of Medical Research (ICMR), Ministry of Health & Family Welfare, 2008. Guidelines for the Safety Assessment of Foods Derived from Genetically Engineered Plants, pp. 1e33.

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Further reading Ahuja, V., 2018. Regulation of emerging gene technologies in India. BMC Proc. 12 (Suppl. 8), 5e11. Asian Development Bank e Working Report, 2001. Agricultural Biotechnology, Poverty Reduction and Food Security, p. 213. Canadian Council on Animal Care, 2008. Trends in Animal Use. http://www.ccac.ca/Documents/Publications/Statistics/Survey_2009.pdf. Cantley, M., 2002. Biotechnology in Europe-20 Years’ Experience and Current Strategy in Asian Biotechnology and Development Review, p. 17. Department for Environment, Food and Rural Affairs (DEFRA), 2004. Policy on the Use of Animals in Research. http://www.defra.gov.uk/science/ documents/Defra-Policy_%20On_Use_Of_Animals_In_Research.pdf. Department of Biotechnology, Ministry of Science and Technology, Govt. of India, 1990. Recombinant DNA Safety Guidelines and Regulation, pp. 1e10. Department of Biotechnology, Ministry of Science and Technology, Govt. of India, 1998. Revised Guidelines for Research in Transgenic Plants, pp. 1e10. Dyck, M.K., Lacroix, D., Pothier, F., Sirard, M.A., 2003. Making recombinant proteins in animals - different systems, different applications. Trends Biotechnol. 21, 394e399. Einsiedel, E.F., Ross, H., 2002. Animal spare parts? A Canadian public consultation on xenotransplantation. Sci. Eng. Ethics 8, 579e591. M/s Ernst & Young and Freehills, 2001. Australian Biotechnology Report 2001, p. 80. Fenwick, N., Griffin, G., Gauthier, C., 2009. The welfare of animals used in science: how the “Three Rs” ethic guides improvements. Can. Vet. J. 50, 523e530. Food and Drug Administration (FDA) report, 2010. US Environmental Assessment for AquAdvantageÒ Salmon. http://www.fda.gov/downloads/ AdvisoryCommittees/CommitteesMeetingMaterials/VeterinaryMedicineAdvisoryCommittee/UCM224760.pdf. Food and Drug Administration, U.S. Department of Health and Human Services, 2017. Guidance for Industry: Regulation of Intentionally Altered Genomic DNA in Animals (Revision of Guidance #187, “Regulation of Genetically Engineered Animals,”), pp. 1e30. Frederick, R.J., 2003. Prelude to building a framework for implementing biosafety. In: Mclean, M.A. (Ed.), A Framework for Biosafety Implementation: Report of a Meeting, p. 35. Gallagher, H., 2018. Organisms obtained by mutagenesis are GMOs and are, in principle, subject to the obligations laid down by the GMO Directive. In: Court of Justice of the European Union PRESS RELEASE No 111/18 case reference OJ 2001 L106, P1-39. www.curia.europa.eu. Gaskell, G., Allum, N., Stares, S., 2003. Europeans and Biotechnology in 2002: Eurobarometer 58.0. Directorate General Press and Communication, Public Opinion Analysis Unit. Gauthier, C., 2004. Overview and analysis of animal use in North America. ATLA 32 (Suppl. 1A), 275e285. Gayatri, G., 2012. Biotech regulatory approval process in India. Int. J. Pharmaceut. Sci. Rev. Res. 13 (1), 135e137. Ghosh, P.K., Ramanaiah, T.V., 2000. Indian rules, regulations and proceedures for handling transgenic plants. J. Sci. Ind. Res. 59, 114e120. Holt, W.V., Pickard, A.R., Prather, R.S., 2004. Wildlife conservation and reproductive cloning. Reproduction 127, 317e324. HSE, 2014. The SACGM Compendium of Guidance, Part 5: Genetic Modification of Animal, pp. 1e26 (The Genetically Modified Organisms Regulations 2014). www.hse.gov.uk/biosafety/gmo/acgm/acgmcomp. Ingrassia, A., Manzella, D., Martyniuk, E., 2005. The legal framework for the management of animal genetic resources. In: FAO Legislative Study, 89, pp. 1e157. Laible, G., 2009. Enhancing livestock through genetic engineering e recent advances and future prospects. Comp. Immunol. Microb. 32, 123e127. Lassen, J., Gjerris, M., Sandoe, P., 2006. After Dolly e ethical limits to the use of biotechnology on farm animals. Theriogenology 65, 992e1004. Lee, B.C., Kim, M.K., Jang, G., et al., 2005. Dogs cloned from adult somatic cells. Nature 436, 641. Logan, J.S., Sharma, A., 1999. Potential use of genetically modified pigs as organ donors for transplantation into humans. Clin. Exp. Pharmacol. 26, 1020e1025. MacArthur, J.A., Potter, M., Harding, E., 2006. The welfare implications of animal breeding and breeding technologies in commercial agriculture. Livest. Sci. 103, 270e281. Macnaghten, P., 2004. Animals in their nature: a case study of public attitudes to animals, genetic modification and nature. Sociology 38, 533e551. Madkour, M.A., 2001. Overview of Biosafety Status in Egypt in Developing and Harmonising Biosafety Regulations for Countries in West Asia and North Africa. In: Baum, M., et al. (Eds.), p. 45. Miller, J.C., Holmes, M.C., Wang, J., et al., 2007. An improved zinc-finger nuclease technology architecture for highly specific genome editing. Nat. Biotechnol. 25, 778e785. Ministry of Environment, Forest & Climate Change, Govt. of India and biotechnology Consortium India Limited, New Delhi, 2015. Regulatory Framework for Genetically Engineered (GE) Plants in India, pp. 1e16. Ministry of Environment, Forest and Climate Change (MoEFCC), 1986. The Environment (Protection) Act, pp. 1e11. Ministry of Environment, Forest and Climate Change (MoEFCC), 1989. Rules for Manufacture, Use/import/export & Storage of Hazardous Microorganisms/genetically Engineered Organisms or Cells. G.S.R. 1037(E). http://www.oecd.org. Oritz, G., Elizabeth, S., 2004. Beyond welfare: animal integrity, animal dignity, and genetic engineering. Ethics Environ. 9, 94e120. Ormandy, E.H., 2010. The Use of Genetically-Engineered Animals in Science. A Report from the Third Genome BC Knowledge Translation Workshop; Vancouver BC. http://www.genomebc.ca/portfolio/genomics-and-society/events/knowledge-translation-workshops/genetically-engineered-animals/ Last.

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Ormandy, E.H., Schuppli, C.A., Weary, D.M., 2009. Worldwide trends in the use of animals in research: the contribution of genetically modified animal models. ATLA 37, 63e68. Ormandy, E.H., Dale, J., Griffin, G., 2011. Genetic engineering of animals: ethical issues, including welfare concerns. Can. Vet. J. 52 (5), 544e550. Osborne, N., Jackson, I., Cox, D., et al., 2009. Sharing and Archiving of Genetically Altered Mice: Opportunities for Reduction and Refinement. A Report of the RSPCA Resource Sharing Working Group (RSWG) from: http://www.nc3rs.org.uk/downloaddoc.asp?id¼857. Rao, S.R., 2002a. Industry feature-time for action. Times Global Journal on Indian Biote©chnology. 30e33. Rao, S.R., 2002b. Transgenic plants: biosafety and people. J. Plant Biol. 29 (1), 1e8. Rao, S.R., 2003. Status of regulation of genetically engineered products in selected countries. Asian Biotechnol. Dev. Rev. 11, 1e38. Rao, S.R., 2017. Regulations and Guidelines for Recombinant DNA Research and Biocontainment. Department of Biotechnology, Ministry of Science & Technology, Government of India, pp. 1e148. Reynaldo, E., Cruz, de la, 2002. Status of Agribiotechnology in the Philippines. Asian Biotechnology and Development Review, p. 22. Robinson, V., Morton, D.B., Anderson, D., et al., 2003. Refinement and Reduction in Production of Genetically Modified Mice. Sixth Report of the BVAAWF/FRAME/RSPCA/UFAW Joint Working Group on Refinement. http://www.arsal.ro/wp-content/uploads/members/13.%20Refinement% 20and%20reduction%20in%20production%20of%20genetically%20modified%20mice.pdf. Rogers, A.D., 2007. Evolution and biodiversity of Antarctic organisms: a molecular perspective. Philos. Trans. R. Soc. Lond. B Biol. Sci. 362 (1488), 2191e2214. Rollin, B.E., 2003. On telos and genetic-engineering. In: Armstrong, S.J., Botzler, R.G. (Eds.), Animal Ethics Reader. Routledge, London, UK, pp. 342e350. Rudmann, D.G., Durham, S.K., 1999. Utilization of genetically altered animals in the pharmaceutical industry. Toxicol. Pathol. 27, 111e114. Schicktanz, S., 2006. Ethical considerations of the human-animal-relationship under conditions of asymmetry and ambivalence. J. Agric. Environ. Ethics 19, 7e16. Schuppli, C.A., Weary, D.M., 2010. Attitudes towards the use of genetically-modified animals in research. Publ. Understand. Sci. 19, 686e697. Shin, T., Kraemer, D., Pryor, J., et al., 2002. Cell biology: a cat cloned by nuclear transplantation. Nature 415, 859. Tabei, Y., 1999. Addressing Public Acceptance Issues for Biotechnology: Experience from Japan in Managing Agricultural Biotechnology. In: Cohen, J.I. (Ed.), p. 174. Traynor, L.P., 1999. Biosafety Management: Key to the Environmentally Responsible Use of Biotechnology in Managing Agricultural Biotechnology. In: Cohen, J.I. (Ed.), p. 155. Trigo, E.E., AchaInter, P.N., 1988. Guidelines for the Use and Safety of Genetic Engineering Techniques or Recombinant DNA Technology. American Institute for cooperation on agriculture, Pan-American Health Organization/World Health Organization, Organization of American States, International Office of Epizootics, pp. 1e104. Tripathi, K.K., Ahuja, V., 2011. Guidelines and Handbook for Institutional Biosafety Committees (IBSCs). Department of Biotechnology, Govt. of India & Biotech Consortium India Limited, New Delhi, pp. 1e120. Trump, D.J., 2019. Executive Order on Modernizing the Regulatory Framework for Agricultural Biotechnology Products. The White House, pp. 1e4. United States Department of Energy Genome Projects, 2009. Cloning Fact Sheet. Veerhoog, H., 1992. The concept of intrinsic value and transgenic animals. J. Agric. Ethics 147e160. Verbeek, J.S., 1997. Scientific applications of transgenic mouse models. In: Van Zutphen, L.F.M., Van Der Meer, M. (Eds.), Welfare Aspects of Transgenic Animals - Proceedings EC Workshop. Springer-Verlag, Berlin, pp. 1e17. Weaver, S.A., Morris, M.C., 2005. Risks associated with genetic modification: an annotated bibliography of peer reviewed natural science publications. J. Agric. Environ. Ethics 18, 157e189. Wells, D.J., 2010. Genetically modified animals and pharmacological research. In: Cunningham, F., Elliot, J., Lees, P. (Eds.), Comparative and Veterinary Pharmacology. Springer, pp. 213e226. Wells, D.J., Playle, L.C., Enser, W.E.J., et al., 2006. Assessing the welfare of genetically altered mice. Lab. Anim. 40, 111e114. West, C., 2006. Economic and ethics in the genetic engineering of animals. Harv. J. Law Technol. 19, 413e442. Wilmut, I., Beaujean, N., de Sousa, P.A., et al., 2002. Somatic cell nuclear transfer. Nature 419, 583e587. World Health Organization (OIE), 2010. Definition of Animal Welfare, Glossary. Terrestrial Animal Health Code, p. 1014. 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Chapter 16

Silkworm genomics: current status and limitations Manjunatha H. Boregowda Department of Studies in Sericulture Science, University of Mysore, Mysore, Karnataka, India

16.1 Introduction The silkworm, Bombyx mori L. (Lepidoptera: Bombycidae), is an economically important and truly domesticated insect widely distributed in many regions, especially China, India, Japan, and Korea. According to archaeological evidence and demographic history involving gene flow of domesticated species, the starting time of silkworm domestication as estimated is w7500 years ago. The time of domestication termination is 3984 years ago (Yang et al., 2014), but phylogenetic analysis reveals domestication of silkworm supposed to occur w4100 years ago (Sun et al., 2012). However, the phenomenon of estimation is well consistent with the domestication time of rice (w7500 years), maize (w7500 years), and most livestock (8000e10,000 years ago (Yang et al., 2014)). Over the years, the silkworms have been reared exclusively for the production of silk yarn and considered as a model insect for various fields of biology, which includes genetics, biochemistry, physiology, developmental biology, neurobiology, pathology, and molecular biology, because of its short life cycle, larger body size, comfortable mass rearing, and laboratory culture. Currently, it is not only used for the production of medically and industrially important biomaterials and recombinant proteins but also considered as a potent model organism in the field of biomedical research to assess the toxicity and efficacy of novel medically important compounds/drugs (Likhith Gowda and Manjunatha, 2019). By and large, the majority of the silk moth species belong to the family of Bombycidae, while few are grouped in the family Saturniidae of the superfamily Bombycoidea in the order Lepidoptera. It comprises 160,000 species, while most of them are agriculturally important pests. As a consequence, B. mori is considered as central model species among Lepidopteron and the first fully sequenced Lepidoptera (Alencon et al., 2010), whose genome sequence is established as a valuable reference for comparative genomics and genetics toward understanding insect diversity and other allied aspects. The comparative genomic analysis, considering a reference genome from a model species - B. mori, has gained significance at a time when competition for food among humans and insects is becoming a critical challenge for a fast-rising human population, to accelerate innovative research in pest management. But the limitation posed during comparative analysis is that does the holocentric nature of the multiple chromosomes in Lepidoptera with a haploid set of 28 chromosomes (n ¼ 28) favors scrambling of gene order and masks microsynteny relationships. Because the Bombyx genome has the highest level of repetitive sequences (43.6%) compared to all insect genomes studied [versus Apis mellifera with just 1% (Alencon et al., 2010). Since the silkworm genome sequence has high significance as a reference, might be with some limitations, not only in Lepidopteran but also other allied species comparative genomics, we have computed information’s available to date and presented in this chapter.

16.2 Genomic basis of the demographic history of the domesticated silkworm, Bombyx mori B. mori has 28 (n) chromosomes, known to derived from the wild silk moth, B. mandarina (n ¼ 27) - due to primary nondisjunction in which one of the chromosomes might have split into two during the process of domestication (Murakami and Imai, 1974). But, the limitation in the knowledge of its origin still exists, because, one of the wild silkworms exploited,

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is from China and far eastern Russia called Chinese B. mandarina, and other from Japan and South Korea called Japanese B. mandarina. During the period of domestication, bidirectional gene flow occurs, which eventually creates more than 1000 inbred strains of silkworms that are distributed worldwide today. The bottleneck limitation for comparative analysis among these two variants and domesticated silkworm strains is due to the lack of genome sequence data for B. mandarina. Still, domesticated strains are clearly genetically differentiated from the wild ones, with w83% of the variation observed in the wild silkworm. According to nuclear genome data, the domesticated silkworm lost approximately 17% of nucleotide diversity measured by q relative to wild silkworm (Xia et al., 2009). Whereas genetic evidence and coalescent simulation methods reveal that the domesticated silkworm lost 33%e49% of nucleotide diversity relative to wild silkworm, which might attribute to a historical bottleneck during its domestication (Guo et al., 2011). This large gap in the estimation of nucleotide diversity is due to two factors; the nucleotide diversity in the domesticated silkworm is found to be at a very similar level measured as q between the classic sequencing data and Solexa resequencing data, which is greatly underestimated, and the other one is lack of sophisticated statistical methods employed then to study the demographic history of the silkworm. In later days, these limitations have been overcome with the use of coalescent simulation and the latest Approximate Bayesian Computation (ABC) methods for assuming different demographic models in the Bayesian framework to infer the evolutionary history of model species (Beaumont et al., 2002). For arriving this point, phylogeny and evolutionary history of silkworm are used, which is based on mitochondrial and nuclear loci sequences (Yang et al., 2014). Even with the availability of copious genomic data, it shall be better to use the inferred demographic history of the domesticated silkworm by comparing the whole-genome resequencing data of the wild silkworm, B. mandarina. The hypothesis inferred to describe the demographic history and the gene flow between B. mori and its wild relative, B. mandarina is based on three possible reasons; first, the overlapping geographic regions between the domesticated and wild silkworm populations might facilitate the gene flow. Wild silkworm is not only widely distributed in the regions of Jiangsu and Zhejiang provinces but also can be found in Anhui and Shandong provinces in China. Interestingly, these areas are also the main regions of sericulture; second, lack of complete reproductive isolation that leads to gene flow between the populations. In fact, the domesticated and wild silkworms have not formed complete reproductive isolation, wherein they have the ability to interbreed with each other, and this facilitated the gene exchange between these two species; third, during silkworm domestication, the environmental factors may also provide some opportunities for the gene flow between the populations. The initial rearing conditions of the domesticated silkworm may be very simple and open compared with present-day’s rearing environments. This might also increase the opportunities of contact and hybridization between the domesticated and wild silkworms. Besides these factors, economic expansion and urbanization might also affect gene flow. Quick economic expansion and intensive urbanization are the common causes of the habitat fragmentation and may eventually reduce the gene flow in wildlife populations between different fragmented landscapes. Thus, economic expansion and urbanization could be the reasons of why gene flow occurred at the period of bottleneck rather than at the recent. Nevertheless, it is obvious that gene flow is ubiquitous between domesticated species and their wild relatives, which play an important role in the domestication of crops and animals. It is interesting to note that bidirectional gene migration between the domesticated and wild silkworm populations has occurred rather than the unidirectional gene flow. However, the gene flow direction from the domesticated silkworm to wild silkworm might be caused by the situation that the domesticated silkworms are discarded together with their excrement into the natural environment. The gene flow from wild silkworm to the domesticated silkworms might be due to wild silkworms are introduced by breeders, as an important genetic source, to hybrid with the domesticated silkworm to produce desirable strains. Still, as more and more population genomic data become available, and application of new statistical methods might uncover the long-standing questions about the role of gene flow in species formation.

16.3 Cytogenetics of the silkworm, Bombyx mori The discovery of business-related benefits of silk in the 19th century brought silkworm to the limelight in Japan. Since then, the silkworm has been exploited in many basic genetic investigations to improve the quality and quantity of silk. Gradually, silk moths have emerged as one of the favorite models for classical genetic studies due to their larger body mass and easy to rear large population under controlled condition. Consequently, hundreds of different strains/races/breeds have been developed using traditional/conventional breeding strategies. But, genome-based technology for the development of new silkworm strains of the desired characteristic needs to be explored. The silkworm, B. mori is one of the model organisms in genetic research next to the fruit fly, Drosophila melanogaster with >3000 known strains (Yamamoto, 2000) and >400 mutations that correspond to w230 mapped genes or loci. But, the karyomorph of this insect has not clear until recent years, except the chromosome number n ¼ 28 (Kawaguchi, 1928)

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and 2n ¼ 56 (Kawamura, 1979) are known. Over a long period, there is no common rule for chromosome recognition, but a pattern of sex chromosomes are identified as heterogametic (ZW) in female and homogametic (ZZ) male (Traut et al., 1999; Sahara et al., 2003). Bombyx possesses a small and high number of holokinetic chromosomes, which are typical features of other moths and butterflies (Lepidoptera). Since Bombyx chromosomes lack primary constriction, uniform in size during mitotic metaphase (Fig. 16.1A) and no discrete banding pattern, it has become a difficult task to differentiate chromosomes not only for its precise identification but also for genome mapping. Subsequently, conditions are better with meiotic chromosomes wherein the pachytene stage chromosomes (Fig. 16.1B) are having an extended length (thread-like structure), pairwise synopsis, and display chromomere patterns (Traut, 1976). With the application of fluorescence in situ hybridization (FISH) using bacterial artificial chromosomes (BACs) probes on meiotic chromosome complements, each of the 28 bivalents of the B. mori karyotype can easily be recognizable by its labeling pattern and chromosome-specific signals. Now, using these BACs probes, a complete karyotype of B. mori can be identified individually with ease (for more details, see Yashido et al., 2005) and construct an accurate physical map.

16.4 Silkworm genomics Whole genetic information or a total number of genes present in a cell of an organism is known as the genome; the term was first coined by Hans Winker in 1920 (Yadav, 2007) drawing letters “Gen” from the word “gene” and “ome” from “chromosome.” The study of its molecular organization and its information as a function is referred to as “Genomics”- the word proposed by Thomas H. Roderick in 1986 (Kuska, 1998). In principle, the word Genomics was proposed by him in an international meeting in Bethesda, while discussing the possibility of mapping the whole human genome and starting a new genome-oriented scientific journal. The word genomics was not just the objective of the journal, but it means GENOM-ICS and state as an activity, a new way of thinking about biology. It encompasses sequencing, mapping, and new technologies, besides comparative analysis of genomes of various species, their evolution, and how they related to each other. Today, the word genomics has become standard terminology in life science research. Genome sequence information is highly valuable in understanding the blueprint of an organism, and finding gene coding sequences in the sequenced DNA is a real challenge. The next priority after genome sequencing is genome annotation. Identifying genes and assigning them a function in a genome sequence provides necessary means to determine biological function. The high-throughput genome annotation uses a combination of comparative and noncomparative data to identify protein-coding genes in the genome sequence. Thus, silkworm genome information not only makes a strong impact on improving sericulture but also facilitates the development of new methods for pest control. The first complete genome sequence of 1830 kb is from a free-living organism Haemophilus influenzae in 1995 that eventually served as a standard method for sequencing other microbial genomes, Mycoplasma genitalium and Mycobacterium tuberculosis. Since then, numerous Archaeal, bacterial and eukaryotic genomes, including those of model organisms such as Saccharomyces cerevisiae, Caenorhabditis elegans, Drosophila melanogaster, Arabidopsis thaliana, mouse, rat and sea urchin are sequenced. Subsequently, the first finished human genome sequence was published in 2004, but a year later, it is considered complete with the entire genome sequence. Prior to it, a draft human genome sequence published concurrently by a publicdWellcome Trust Sanger Institute (International Human Genome Sequencing

FIGURE 16.1

Chromosome complements of Bombyx mori. (A) Metaphase, (B) Pachytene chromosomes.

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Consortium, 2001) and privatedCelera Genomics (Venter et al., 2001) groups with 90% coverage, which was not a complete final sequence. The International Human Genome Sequencing Consortium managed to acquire a finished sequence of at least 95% of the euchromatin, part of the genome in which most of the genes are located. The progress in genomic research from bacteria (prokaryotes) to human (eukaryotes) led to the initiation of genome sequencing in B. mori, second only to the fruit fly as a model insect for genetics, hoping that it might bridge the gap between the fly (180 Mb) and the human (3000 Mb) as the haploid genome size of the silkworm (432 Mb) is 2.4 times greater than that of D. melanogaster and one-seventh that of the human genome. Therefore, an ambitious silkworm genome project was initiated by China, Japan, France, and other countries (Xia et al., 2004; Mita et al., 2004) to establish a basic resource for comprehensive genome analysis. Consequently, a large number of insect genome projects are initiated and the whole-genome sequence for >25 insects is completed, and sequencing of many more insects’ is in progress under the i5K project (www.arthropodgenomes.org/wiki/i5K). With the advent of sequencing facilities and genome informatics, whole-genome sequencing analyses are carried out not only in viral and microbial genomes but also in multicellular organisms, including D. melanogaster (Myers et al., 2000), A. gambiae (Holt et al., 2002) and human (International Human Genome Sequencing Consortium, 2001). The same sequencing strategy was adopted for B. mori, initially, by both Japanese (Mita et al., 2004) and Chinese (Xia et al., 2004) research groups independently and subsequently, advanced genome technology employed to produce more accurate genome sequence assembly. Keeping the substantial generation of B. mori genome sequence data for the past 2 decades in consideration, in this chapter, we present consolidated information for a better understanding of the silkworm genome and facilitating comparative analysis among insects.

16.5 Silkworm genome programs From inception (1994) until the production of a high-quality silkworm genome sequence (2019), the silkworm genome research performed is distinctly categorized as follows Phase I: Draft genome assembly - performed concurrently but independently by o Japanese group (Mita et al., 2004) - produced 3  genome assembly. o Chinese group (Xia et al., 2004) - generated 6  genome assembly. Phase II: Integrated or finishing genome assembly - performed jointly by o The International Silkworm Genome Consortium, (2008) - integrated 8.5  genome assembly. Phase III: Final and high-quality new genome assembly o High-quality genome assembly (Kawamoto et al., 2019). In the year 1994, which shall be treated as phase I, domesticated silkworm, B. mori genome project was initiated as a silkworm genome research program (SGP) at the National Institute of Sericultural and Entomological Science (NISES), which later coordinated by the Insect Genome Research Team. As a result, a comprehensive linkage map of the silkworm genome was constructed and released as “BombMap” in 1996. Subsequently, a database “SilkBase” was constructed comprising partial cDNA sequence information and made accessible to the public in 1999. Currently, all the information is merged into a core database, KAIKObase. Concomitantly, Southwestern University, in collaboration with Beijing Genomics Institute, has undertaken the silkworm genome project in China. Both Japanese (Mita et al., 2004) and Chinese (Xia et al., 2004) groups published the silkworm genome sequence data at the same time but independently. Due to low coverage in these two projects, the genome sequence data, in some regions, have insufficient sequence information and warranted integration of these two data sets with the additional sequence information for accurate gene annotation and genome sequence data. To meet this challenge, in phase II, the National Institute of Agrobiological Sciences (NIAS) in Japan and the Southwest University in Chongqing City, China have made a joint research agreement on March 24, 2006, in order to integrate both the silkworm genome sequences and constituted silkworm genome consortium, including related researchers across the globe to aid structural and functional analysis of the silkworm genome. Consequently, the silkworm genome resequencing project performed large scale sequencing of 40 silkworms and wild silkworms with different geographical regions, physiological and economic traits to discover target genes and genomic regions associated with domestic and artificial selection (Xia et al., 2009). Thereafter, a large section of researchers throughout the world have contributed greatly toward the silkworm genome, which has been summarized in the following sections.

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16.6 Silkworm genome sequence 16.6.1 Phase I: draft genome sequence 16.6.1.1 Japanese 3  - genome assembly 16.6.1.1.1 Genome sequence and assembly methods For genome sequencing, genomic DNA is derived from the posterior silk glands of day-3 fifth instar larvae of silkworm strain p50T (Daizo). The genomic DNA is fragmented using the HydroShear process (GeneMachines Inc., USA); fragments of 2e3 kb and 7e10 kb are ligated into a pUC18 and pTWV228 plasmid vectors respectively. The ligated DNA samples are introduced into E. coli by electroporation that resulted in two genomic libraries. Sequencing is carried out from both the ends of plasmid DNA using ABI-3700 capillary sequencer and BigDye Terminator v3.1 Cycle Sequencing Kit (Applied biosystems) in one center and MegaBACE4000 capillary sequencer and DYEnamic ET Terminator Cycle Sequencing Kit (Amersham Biosciences) at another center. The sequence data are assembled using a software program RAMEN assembler and deposited in DNA Data Bank of Japan (DDBJ) under accession numbers BAAB01000001 to BAAB01213289.

16.6.1.2 Genome sequence assembly The Japanese group has generated threefold Whole-Genome Shotgun (WGS) sequence with 213,289 sequence contigs and 49,345 scaffolds of 10 kb in an average length. The estimated total scaffolds length is 514 Mb, including gaps and 387 Mb without gaps, which comprise the largest sequence contigs and scaffolds of 19,243 bp and 224,537 bp, respectively. However, the estimated genome size based on DNA reassociation kinetics is 530 Mb (Gage, 1974), and the value, as determined using flow cytometry, is 450e493 Mb as against 514 Mb from WGS. So, the genome size of the silkworm, if considered as 530 Mb, then almost 97% of the genome is organized in scaffolds, of which 75% has been sequenced. However, the predicted sequence coverage is to about 86% with gaps, and 72.9% without gaps. As per the WGS, the G þ C content is 32.54% in the whole genome and 674,490 total mate pairs in scaffolds (for more details see Mita et al., 2004). The GC rich regions have a higher density of transposable elements (TEs) and long interspersed nuclear elements (LINEs) in particular, which are quite different from what is reported for the human and mouse genomes (Xia et al., 2004). Due to the presence of high-copy repetitive sequence and holocentric nature of Bombyx (Lepidopteran) chromosomes, the accuracy in assembly and the precise genome coverage is estimated aligning sequence contigs and scaffolds based on five BAC clones sequenced. A comparative analysis of reference and aligned sequence contigs explicit a total of 0.08% sequence error and 2% misassembly rate, which is reasonable for threefold redundancy.

16.6.1.3 Detecting genes in the WGS Toward validation of WGS data by BLAST search, 50 characteristic genes with complete coding sequences (CDSs) retrieved from the public database are used as reference genes (for more details see Mita et al., 2004). Of the 50 genes, 32 genes are having 90% coverage and two genes showed 50% coverage indicating the possibility of detecting 60% of genes in WGS sequence contigs that open ample scope for the discovery of novel genes. Further, 11,202 nonredundant ESTs available in SilkBase are used for homology search against WGS using BLASTN, that explicit more than 50% coverage, indicating the validity of the WGS data for detecting a large section of Bombyx genes. Moreover, the most prominent feature of the B. mori genome is the abundance of TEs, covering 12% of the genome with an average length of