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AdvancesinAnimalGenomics
Edited by
Sukanta Mondal
Ram Lakhan Singh

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Dedicated to our beloved daughters
Anoushka (Daughter of Mrs. Shrabanti Mondal and Dr. Sukanta
Mondal), for making everything worthwhile and cheerfully
toleratingthe manyhoursof absenceinvolvedinwritingthisbook
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

Contributors
Sarah Afreen, DBT-BIF Facility, Department of Bio-
technology, Maharani Lakshmi Ammanni College for
Women, Bengaluru, Karnataka, India
Kunal Ankola, Department of Studies in Sericulture Sci-
ence, University of Mysore, Mysore, Karnataka, India
R.L. Babu, Department of Bioinformatics and Bio-
technology, Karnataka State Akkamahadevi Women’s
University, Jnanashakthi Campus, Vijayapura, Karna-
taka, 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, Govern-
ment of India, New Delhi, Delhi, India
Shivaleela Biradar, Department of Bioinformatics and
Biotechnology, Karnataka State Akkamahadevi
Women’s University, Jnanashakthi Campus, Vijaya-
pura, Karnataka, India
Manjunatha H. Boregowda, Department of Studies in
Sericulture Science, University of Mysore, Mysore,
Karnataka, India
Shane Carrion, Department of Animal Sciences, Wash-
ington State University, Pullman, WA, United States
D.N. Das, Genetics Laboratory, Dairy Production Section,
ICAR-National Dairy Research Institute (SRS),
Southern Regional Station, Bangalore, Karnataka, 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
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, Wash-
ington 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&Bio-
technology, 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
LikhithGowda Mahadevegowda, Department of Studies
in Sericulture Science, University of Mysore, Mysore,
Karnataka, India
Dhruba Malakar, Animal Biotechnology Centre, National
Dairy Research Institute, Karnal, Haryana, India
Hruda Nanda Malik, Animal Biotechnology Centre, Na-
tional 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
xv

Sukanta Mondal, ICAR-National Institute of Animal
Nutrition and Physiology, Bangalore, Karnataka, India
S. Mondal, ICAR-National Institute of Animal Nutrition
and Physiology, Bangalore, Karnataka, India
Nibedita Naha, Biochemistry Division, ICMReNational
Institute of Occupational Health (NIOH), Ahmedabad,
Gujarat, India
S. Nandi, ICAR-National Institute of Animal Nutrition and
Physiology, Bangalore, Karnataka, 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, Karna-
taka, 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, GreaterNoida, 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
Shikha, Department of Environmental Science, Babasaheb
Bhimrao Ambedkar University, Lucknow, Uttar Pra-
desh, India
Pankaj Singh, Department of Biotechnology, Dr. Ram-
manohar Lohia Avadh University, Ayodhya, Uttar
Pradesh, India
Pradeep Kumar Singh, Department of Biochemistry, Dr.
Rammanohar Lohia Avadh University, Ayodhya, Uttar
Pradesh, 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, Mad-
hya 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: Nilamber-Pitamber
University, Medininagar, Palamu, Jharkhand, India
Neeraj Sinha, Centre of Biomedical Research, SGPGIMS-
Campus, 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, Wash-
ington State University, Pullman, WA, United States
xviContributors

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 de-
mand, 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, andﬁber 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 tran-
scriptome 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 chal-
lenges 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 genomics-
assisted breeding approaches in diverse animal genotypes with contrasting yield and abiotic stress tolerance traits. Un-
fortunately, 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 mo-
lecular 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.
xvii

The contributors to the book are internationally recognized experts in theirﬁeld, 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 summaryhas been presented at the beginning of each chapter to highlight the important concepts discussed in the
chapter.
The contentsof each chapter list the main topics included in that chapter.
Tables andﬁguresdispersed throughout the chapters enable easy understanding of the concepts discussed.
The bibliographyat 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 introductiontraces the brief introduction, scope, and applications of animal genomics for sustainable livestock
production.
From gene to genomics: tools for improvement of animalsdescribe 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 diseasesinvolves 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 animalsdeals 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 transcriptomicsdeals with the transcriptional landscape: regulatory
RNAs and its analysis, transcriptome sequencing, and future perspectives.
Targeted genome editing: a new era in molecular biologyhighlights 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 improvementdemonstrates 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 challengescover 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 breedingfocus 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), Allo-
zyme markers, Mitochondrial DNA (mtDNA), DNA Barcoding markers, Marker-assisted selection (MAS) and its
application.
Genomic selection: a molecular tool for genetic improvement in livestockinvolves 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 therapyillustrates 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 healthcovers quantum dot nanoparticles, carbon-based nanoparticles,
dendrimers nanoparticles, liposomes nanoparticles, metal and metal oxides nanoparticles, polymeric nanoparticles, etc.
Cell Signaling and apoptosis in animalsinvolves 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.
xviiiPreface

Molecular network for management of neurodegenerative diseases and its translational importance using
animal biotechnology as a tool in preclinical studiesinvolves 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 genomicsdemonstrates genetic engineering technology/genomics for animal husbandry
application, genetic engineering technology/genomics for animal husbandry application, genomics versus policy, regu-
lations 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 limitationsdiscuss 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 approachesillustrates 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 barcodingcovers 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
Prefacexix

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 forﬁnishing 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
xxi

Chapter 1
Introduction
Pankaj Singh
1
, Sukanta Mondal
2
and Ram Lakhan Singh
3,*
1
Department of Biotechnology, Dr. Rammanohar Lohia Avadh University, Ayodhya, Uttar Pradesh, India;
2
ICAR-National Institute of Animal
Nutrition and Physiology, Bangalore, Karnataka, India;
3
Department of Biochemistry, Dr. Rammanohar Lohia Avadh University, Ayodhya, Uttar
Pradesh, 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:
lStudy of organization and expression of the genome as speciﬁc traits.
lCharacterization of the complete genome rather than one gene at a time with the application of new technologies.
lSequencing of mainly livestock and poultry genomes to understand the functional genomics.
The discovery of new techniques and technologies in theﬁeld 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; prote-
omics 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 re-
searchers to move into large-scale gene expression studies. The human genome project was completed in 2003, with a cost
of approximately $18 million. The keyﬁndings 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. 1

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 1.1The central dogma of omics science, which integrates information from structural genomics to transcriptomics, proteomics, and
phenomics.
FIGURE 1.2Schematic overview of network analysis of the genome to understand the integration of information of transcriptome, proteome,
metabolome, and how these interactions determine biological functions.
2Advances in Animal Genomics

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 1.3Role of epigenetic modiﬁcation in the development of impaired physiological functions.
IntroductionChapter | 13

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 in-
dividuals 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), micro-
satellites, 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 1.4Steps involved in the metagenomics for the analysis of metagenome.
FIGURE 1.5Factors and role of genetic polymorphism in the determination of pharmacogenomics properties in humans.
4Advances in Animal Genomics

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 oligo-
nucleotides (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 byLitt 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 tech-
nology. Constructed transgene must be integrated to the host genome and should be stable so that they pass on to sub-
sequent 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
IntroductionChapter | 15

TABLE 1.1Advantages 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
-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)-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
-Can not be used across species
-reproducibility low
-Dominant markers
-No information of probe or primer
Simple sequence repeat (SSR)-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
6Advances in Animal Genomics

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 addi-
tional 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.
IntroductionChapter | 17

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 ge-
nomics 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 animalﬁtness, 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, theﬁrst 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 theﬁrst group, which includes multiple ovulation, arti-
ﬁcial 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 charac-
teristics (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 1.6Application of animal genomics.
8Advances in Animal Genomics

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 agriculturalﬁeld, 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 (Uni-
versity of Washington) created theﬁrst 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 bodyﬂuids, 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, andﬁnd 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 dis-
orders, malignancies, neuropsychiatric illnesses, developmental diseases, etc.
Application of gene therapy involves the following steps:
1.Identiﬁcation of the gene that plays a key role in the development of a genetic disorder
2.Determination of the role of its products in disease development
3.Isolation and cloning of the gene
4.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.Disease should be life threatening
2.The gene responsible for the disease has been cloned
3.A precise regulation of the gene should not be required
4.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
IntroductionChapter | 19

(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, andﬁber 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 syntheticﬁber 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 ofStaphylococcus aureus,responsible for the majority of mastitis cases. According to available
reports, theﬁrst transgenic cows have been produced, which are resistant toS. aureusmediated 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, aﬁsh 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 manyﬁelds, 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.
10Advances in Animal Genomics

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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.
12Advances in Animal Genomics

Chapter 2
From gene to genomics: tools for
improvement of animals
Pradeep Kumar Singh
1
, Pankaj Singh
2
, Rajat Pratap Singh
3
and Ram Lakhan Singh
1,4
1
Department of Biochemistry, Dr. Rammanohar Lohia Avadh University, Ayodhya, Uttar Pradesh, India;
2
Department of Biotechnology,
Dr. Rammanohar Lohia Avadh University, Ayodhya, Uttar Pradesh, India;
3
Department of Biotechnology, Guru Ghasidas University, Bilaspur,
Chhattisgarh, India;
4
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 the1953landmark 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 fruitﬂy 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:
lNoncoding regions, called introns, which do not specify amino acids and are removed (spliced) from the mRNA mole-
cule before translation (Fig. 2.1).
lCoding 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 theﬁnal mature mRNA molecule.
lRegulatory 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 bacteriumEscherichia 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. 13

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,
whereasVibrio cholerahas 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.
Nucleus
Mitochondria
Cell Strands
5’
3’
3’
5’
Base pair
Sugar phosphate
backbone
DNA Double helix
Pairs of chromosomes
ina human cell
123 45
6789101112
131415 161718
1920 21 22 23
XY
FIGURE 2.2Chromosomes in human Cell and DNA structure.
FIGURE 2.1A structural gene involves a number of different components.
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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 informationﬂow 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 require-
ment 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 pro-
karyotes. 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 theﬁnal mature mRNA molecule and
Regulatory sequences
Enhancer/
Silencer Promoter5’UTR Open reading frame
Regulatory sequences
3’UTR
Enhancer/
Silencer
Proximal Core Start Stop Terminator
Exon 1 Exon 2 Exon 3
Intron 1Intron 2
Protein coding region
5’cap
Transcription
Pre-mRNA
Post-transcriptional
modification
Translation
Protein
mRNA
Poly A tail
Protein
FIGURE 2.3Structure 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 5
0
cap
and poly-A tail. Finally, mature mRNA translates into the protein product.
From gene to genomics: tools for improvement of animalsChapter | 215

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. 5
0
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
Enhancet/
Silencer 5’UTROpen reading frame Open reading frame3’UTR
Regulatory sequences
Enhancet/
Silencer
Start StartStop Stop Terminator
Transcription
Translation
Protein Protein
Operator Promoter UTR
RBS
Protein coding regionRBS Protein coding region
FIGURE 2.4Structure 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 theﬁnal protein products.
16Advances in Animal Genomics

Transcription of the gene into an mRNA is regulated by the promoter, operator, and enhancer regions. Theﬂanking
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 5
0
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) ofan organism. Every genome contains entireinformation
expected to develop and keep up that life form. In eukaryoticcells, a large portion of DNA ispresent 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
chromosomesﬂuctuates 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.
Gene and Gene related sequences Intergenic DNA
Exons Regulatory sequences
Example: Promoter
Gene related sequences
Genome wide repeats
Examples: Transposan
Introns Gene Fragments Pseudo Genes
Other Intergenic regions
Microsatellite
Minisatellite
Unique Sequences
Eukaryotic Genome
FIGURE 2.5Anatomy 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 inﬂuenzaewas theﬁrst organism whose entire genome was sequenced in 1995 (Fleischmann et al., 1995).
Theﬁrst eukaryotic organism to be sequenced wasSaccharomyces cerevisiaein 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.
18Advances in Animal Genomics

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 tech-
niques 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 single-
stranded 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 5
0
end. This marked substrate is exposed to four separate cleavage
reactions (G, AþG, C, CþT) 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
3
0
position. These modiﬁed nucleotides inhibit the addition of further nucleotides because they lack a 3
0
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
Shotgun Approach Clone Contig Approach
Genomic DNA
Break genomic into smaller
fragment by sonication
Sequencing
Examined for overlaps
Assembled on the basis of sequence overlaps
Break genomic into large
fragment by restriction enzyme
Each fragment is cloned in a suitable host-vector system
Organized mapped large clone contigs
Sequencing of each clone
Assemble sequences of overlapping clones
FIGURE 2.6Genome sequencing approaches: Shotgun and Clone contig approach.
From gene to genomics: tools for improvement of animalsChapter | 219

the nucleotides should be radioactively orﬂuorescently labeled to detect theﬁnal 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 wasﬁnally 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 gene-
related sequences and intergenic DNA sequences. Construction of a genomic DNA library starts with isolation and pu-
riﬁcation 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 aslreplacement vector, cosmid, yeast
artiﬁcial chromosome (YAC), bacterial artiﬁcial chromosome (BAC), or P1 vector is used for cloning of DNA fragments
20Advances in Animal Genomics

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 theﬁrst strand of cDNA by using reverse transcriptase
enzyme.
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 2.7Construction of genomic library.
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 2.8Construction of cDNA library.
From gene to genomics: tools for improvement of animalsChapter | 221

After the synthesis of theﬁrst 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 3ʹend, which
provide 3
0
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 3
0
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). Thisﬁeld 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), func-
tional 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 areﬁrst 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
22Advances in Animal Genomics

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 geno-
mics 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 therole 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 necessaryto 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 hypercholester-
olemia 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 theﬁrst 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 appearin 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, andﬁbronectin 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 arecommon for both organisms suchas 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 asthey have a larger genome. Some regions of the human genome
are highly conserved indogs, cattle, and sheep.
2.4.1.3.4 Horizontal gene transfer
Horizontal or lateral gene transfer is the genetic exchangebetween 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 informationﬂow 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 aspathogenesis, 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 byDrosophila melanogasterfromDrosophila 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 betweenﬁshes (Graham et al., 2008).
Recently, heritable HGT was discovered in humans from the mitochondrial-derived minicircles in theTrypanosoma
cruzi(Hechtetal.,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. HGTevent between the different
domains of life (Archaea, Eubacteria, and Eukaryota) has also been explained; for example, the EubacteriaThermotoga
24Advances in Animal Genomics

maritimaharbor 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 theAgrobacterium 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 toﬁnd 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 (restric-
tion 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 110
ﬁ9
and 510
ﬁ9
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
containw6 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 inﬁnding 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 aDNA 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 chloro-
plast) genome. The amount of DNA present in the genome of a species is called C-value, and the size of the genome
From gene to genomics: tools for improvement of animalsChapter | 225

represents one of the most important parameters to understand the complexity in eukaryotic organisms. Genomic com-
parisons 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 animalhas a mix of traits that could be characterized as simple or
complex (Dunn and Ryan, 2015). The complete genome ofDrosophila melanogastercontains 21 nuclear receptors,
compared to 49 in the human genome (Garcia et al., 2003).Currentgenomicandevolutionaryresearchonnonhuman
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 poly-
morphisms (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 theexact position of a gene on the particular chromosome. It also provides in-
formation 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 progeniesin 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)
26Advances in Animal Genomics

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 rear-
rangements (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; theﬁrst 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 dependson 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).
From gene to genomics: tools for improvement of animalsChapter | 227

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. Theﬁrst 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 mod-
iﬁcations. 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 acety-
lation 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 inE. colito 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 com-
pounds. The lac operon present inE. coliis 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
28Advances in Animal Genomics

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 thelacoperon 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 theﬁrst structural gene of the operon.Trpoperon ofE. coliis an example of an
attenuated operon that encodesﬁve 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 mech-
anisms 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 intro-
gression, 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. Whole-
genome sequencing has made a signiﬁcant contribution to the generation of genomic information (Druet et al., 2014). The
chicken genome was theﬁrst 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 charac-
terizing 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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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 in196

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.
Advances in Animal Genomics.https://doi.org/10.1016/B978-0-12-820595-2.00003-5
Copyright©2021 Elsevier Inc. All rights reserved. 33

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 wereﬁrst 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 undif-
ferentiated 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 mecha-
nisms. The adverse effects of ESCs have also been reported in clinical studies such as tumors and unwanted immune
responses.
FIGURE 3.1Generation of embryonic stem cells from in vitro produced hatched blastocyst.
34Advances in Animal Genomics

3.2.1.2 Adult stem cells
Pluripotent stem cells used for treatment are increasingly restricted in their lineage potential and production of tissue-
speciﬁc 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 wereﬁrst 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
wereﬁrst 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 theﬁeld 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.
Stem cells: a potential regenerative medicine for treatment of diseasesChapter | 335

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 wereﬁltered in a 41mmﬁlter to
remove undigested fat tissue. The cell suspension was centrifuged at 1000 rpm for 10 min, and the pellet was seeded in a
25 cm
2
cultureﬂask 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 cm
2
cultureﬂask at density
210
5
cells/cm
2
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 1106 cells and put
into 2 mL cryovials. The cryovials were kept at 4
ﬂ
C for 4 h andﬁ80
ﬂ
C overnight. Then the cryovials were shifted into a
liquid nitrogen tank for further use. The cryopreserved cells were thawed at 37
ﬂ
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 cultureﬂask and injected
around 10
7
cells/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 3.2Adipose 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.
36Advances in Animal Genomics

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 100mg/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 1000g for 10 min at 25
ﬂ
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% CO
2
incubator at 38.5
ﬂ
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 fruitﬂies,
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.
FIGURE 3.3Directed differentiation of dog adipose tissue-derived Mesenchymal stem cells into Osteocytes, Chondrocytes, Adipocytes, and
Neurocytes.
Stem cells: a potential regenerative medicine for treatment of diseasesChapter | 337

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 theﬁeld 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 theﬁrst 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 Alz-
heimer’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-strengthﬁber 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 fromﬁ20 to 330
ﬂ
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
38Advances in Animal Genomics

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, trans-
plantation 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 emergingﬁeld 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 live-
stock 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 tech-
niques 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 car-
diovascular disease, lungﬁbrosis, spinal cord injury, and bone and cartilage repair. Theﬁrst clinical trial using in vitro-
derived 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 long-
term 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 for-
mation during wound repair (Duschera et al., 2016). According to the International Society of Cellular Therapy (ISCT),
Stem cells: a potential regenerative medicine for treatment of diseasesChapter | 339

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 ne-
crosis factor-ainterleukin-1b, and nitric oxide synthase. MSCs secrete prostaglandin E2 and regulateﬁbrosis, 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 (MCP-
1), 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 factorb-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 immu-
nosuppressive 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 aureuscauses 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).
40Advances in Animal Genomics

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 theﬁrst 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 NDRI-
Karnal, (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 3.4Treatment 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).
Stem cells: a potential regenerative medicine for treatment of diseasesChapter | 341

Exploring the Variety of Random
Documents with Different Content

PSALM CXVI.
1 I love—for Jehovah hears
My voice, my supplications.
2 For He has bent His ear to me,
And throughout my days will I call.
3 The cords of death ringed me round,
And the narrows of Sheol found me,
Distress and trouble did I find.
4 And on the name of Jehovah I called,
"I beseech Thee, Jehovah, deliver my soul."
5 Gracious is Jehovah and righteous,
And our God is compassionate.
6 The keeper of the simple is Jehovah,
I was brought low and He saved me.
7 Return, my soul, to thy rest,
For Jehovah has lavished good on thee.
8 For Thou hast delivered my soul from death,
My eye from tears,
My foot from stumbling.
9 I shall walk before Jehovah in the lands of the living.
10 I believed when I [thus] spake,
"I am greatly afflicted."
11 I said in my agitation,
"All men deceive."
12 What shall I return to Jehovah,
[For] all His goodness lavished on me?
13 The cup of salvations will I lift,
And on the name of Jehovah will I call.
14 My vows will I repay to Jehovah,

Oh! may I [do it] before all His people!
15 Precious in the eyes of Jehovah
Is the death of His favoured ones.
16 I beseech Thee, Jehovah—for I am Thy servant,
I am Thy servant, the son of Thy handmaid,
Thou hast loosed my bonds.
17 To Thee will I offer a sacrifice of thanksgiving,
And on the name of Jehovah will I call.
18 My vows will I repay to Jehovah,
Oh! may I [do it] before all His people!
19 In the courts of the house of Jehovah,
In the midst of thee, Jerusalem.
Hallelujah.
This psalm is intensely individual. "I," "me," or "my" occurs in every
verse but two (vv. 5, 19). The singer is but recently delivered from
some peril, and his song heaves with a ground-swell of emotion
after the storm. Hupfeld takes offence at its "continual alternation of
petition and recognition of the Divine beneficence and deliverance,
or vows of thanksgiving," but surely that very blending is natural to
one just rescued and still panting from his danger. Certain
grammatical forms indicate a late date, and the frequent allusions to
earlier psalms point in the same direction. The words of former
psalmists were part of this singer's mental furniture, and came to his
lips, when he brought his own thanksgivings. Hupfeld thinks it
"strange" that "such a patched-up (zusammengestoppelter) psalm"
has "imposed" upon commentators, who speak of its depth and
tenderness; it is perhaps stranger that its use of older songs has
imposed upon so good a critic and hid these characteristics from
him. Four parts may be discerned, of which the first (vv. 1-4) mainly
describes the psalmist's peril; the second (vv. 5-9), his deliverance;
the third glances back to his alarm and thence draws reasons for his
vow of praise (vv. 10-14); and the fourth bases the same vow on the
remembrance of Jehovah's having loosed his bonds.

The early verses of Psalm xviii. obviously colour the psalmist's
description of his distress. That psalm begins with an expression of
love to Jehovah, which is echoed here, though a different word is
employed. "I love" stands in ver. 1 without an object, just as "I will
call" does in ver. 2, and "I believed" and "I spoke" in ver. 10.
Probably "Thee" has fallen out, which would be the more easy, as
the next word begins with the letter which stands for it in Hebrew.
Cheyne follows Graetz in the conjectural adoption of the same
beginning as in ver. 10, "I am confident." This change necessitates
translating the following "for" as "that," whereas it is plainly to be
taken, like the "for" at the beginning of ver. 2, as causal. Ver. 3 is
moulded on Psalm xviii. 5, with a modification of the metaphors by
the unusual expression "the narrows of Sheol." The word rendered
narrows may be employed simply as = distress or straits, but it is
allowable to take it as picturing that gloomy realm as a confined
gorge, like the throat of a pass, from which the psalmist could find
no escape. He is like a creature caught in the toils of the hunter
Death. The stern rocks of a dark defile have all but closed upon him,
but, like a man from the bottom of a pit, he can send out one cry
before the earth falls in and buries him. He cried to Jehovah, and the
rocks flung his voice heavenwards. Sorrow is meant to drive to God.
When cries become prayers, they are not in vain. The revealed
character of Jehovah is the ground of a desperate man's hope. His
own Name is a plea which Jehovah will certainly honour. Many words
are needless when peril is sore and the suppliant is sure of God. To
name Him and to cry for deliverance are enough. "I beseech Thee"
represents a particle which is used frequently in this psalm, and by
some peculiarities in its use here indicates a late date.
The psalmist does not pause to say definitely that he was delivered,
but breaks into the celebration of the Name on which he had called,
and from which the certainty of an answer followed. Since Jehovah
is gracious, righteous (as strictly adhering to the conditions He has
laid down), and merciful (as condescending in love to lowly and
imperfect men), there can be no doubt how He will deal with trustful
suppliants. The psalmist turns for a moment from his own

experience to sun himself in the great thought of the Name, and
thereby to come into touch with all who share his faith. The cry for
help is wrung out by personal need, but the answer received brings
into fellowship with a great multitude. Jehovah's character leads up
in ver. 6 to a broad truth as to His acts, for it ensures that He cannot
but care for the "simple," whose simplicity lays them open to
assailants, and whose single-hearted adhesion to God appeals
unfailingly to His heart. Happy the man who, like the psalmist, can
give confirmation from his own experience to the broad truths of
God's protection to ingenuous and guileless souls! Each individual
may, if he will, thus narrow to his own use the widest promises, and
put "I" and "me" wherever God has put "whosoever." If he does he
will be able to turn his own experience into universal maxims, and
encourage others to put "whosoever" where his grateful heart has
put "I" and "me."
The deliverance, which is thus the direct result of the Divine
character, and which extends to all the simple, and therefore
included the psalmist, leads to calm repose. The singer does not say
so in cold words, but beautifully wooes his "soul," his sensitive
nature, which had trembled with fear in death's net, to come back to
its rest. The word is in the plural, which may be only another
indication of late date, but is more worthily understood as
expressing the completeness of the repose, which in its fulness is
only found in God, and is made the more deep by contrast with
previous "agitation."
Vv. 8, 9, are quoted from Psalm lvi. 13 with slight variations, the
most significant of which is the change of "light" into "lands." It is
noticeable that the Divine deliverance is thus described as
surpassing the psalmist's petition. He asked, "Deliver my soul." Bare
escape was all that he craved, but he received, not only the
deliverance of his soul from death, but, over and above, his tears
were wiped away by a loving hand, his feet stayed by a strong arm.
God over-answers trustful cries, and does not give the minimum
consistent with safety, but the maximum of which we are capable.

What shall a grateful heart do with such benefits? "I will walk before
Jehovah in the lands of the living," joyously and unconstrainedly (for
so the form of the word "walk" implies), as ever conscious of that
presence which brings blessedness and requires holiness. The paths
appointed may carry the traveller far, but into whatever lands he
goes, he will have the same glad heart within to urge his feet and
the same loving eye above to beam guidance on him.
The third part (vv. 10-14) recurs to the psalmist's mood in his
trouble, and bases on the retrospect of that and of God's mercy the
vow of praise. Ver. 10 may be variously understood. The "speaking"
may be taken as referring to the preceding expressions of trust or
thanksgivings for deliverance. The sentiment would then be that the
psalmist was confident that he should one day thus speak. So
Cheyne; or the rendering may be "I believed in that I spake thus"—
i.e., that he spake those trustful words of ver. 9 was the result of
sheer faith (so Kay). The thing spoken may also be the expressions
which follow, and this seems to yield the most satisfactory meaning.
"Even when I said, I am afflicted and men fail me, I had not lost my
faith." He is re-calling the agitation which shook him, but feels that,
through it all, there was an unshaken centre of rest in God. The
presence of doubt and fear does not prove the absence of trust.
There may live a spark of it, though almost buried below masses of
cold unbelief. What he said was the complaint that he was greatly
afflicted, and the bitter wail that all men deceive or disappoint. He
said so in his agitation (Psalm xxxi. 22). But even in recognising the
folly of trusting in men, he was in some measure trusting God, and
the trust, though tremulous, was rewarded.
Again he hurries on to sing the issues of deliverance, without waiting
to describe it. That little dialogue of the devout soul with itself (vv.
12, 13) goes very deep. It is an illuminative word as to God's
character, an emancipating word as to the true notion of service to
Him, a guiding word as to common life. For it declares that men
honour God most by taking His gifts with recognition of the Giver,
and that the return which He in His love seeks is only our thankful

reception of His mercy. A giver who desires but these results is
surely Love. A religion which consists first in accepting God's gift and
then in praising by lip and life Him who gives banishes the religion of
fear, of barter, of unwelcome restrictions and commands. It is the
exact opposite of the slavery which says, "Thou art an austere man,
reaping where thou didst not sow." It is the religion of which the
initial act is faith, and the continual activity, the appropriation of
God's spiritual gifts. In daily life there would be less despondency
and weakening regrets over vanished blessings, if men were more
careful to take and enjoy thankfully all that God gives. But many of
us have no eyes for other blessings, because some one blessing is
withdrawn or denied. If we treasured all that is given, we should be
richer than most of us are.
In ver. 14 the particle of beseeching is added to "before," a singular
form of expression which seems to imply desire that the psalmist
may come into the temple with his vows. He may have been thinking
of the "sacrificial meal in connection with the peace-offerings." In
any case, blessings received in solitude should impel to public
gratitude. God delivers His suppliants that they may magnify Him
before men.
The last part (vv. 15-19) repeats the refrain of ver. 14, but with a
different setting. Here the singer generalises his own experience,
and finds increase of joy in the thought of the multitude who dwell
safe under the same protection. The more usual form of expression
for the idea in ver. 15 is "their blood is precious" (Psalm lxxii. 14).
The meaning is that the death of God's saints is no trivial thing in
God's eyes, to be lightly permitted. (Compare the contrasted
thought, xliv. 12.) Then, on the basis of that general truth, is built
ver. 16, which begins singularly with the same beseeching word
which has already occurred in vv. 4 and 14. Here it is not followed by
an expressed petition, but is a yearning of desire for continued or
fuller manifestation of God's favour. The largest gifts, most fully
accepted and most thankfully recognised, still leave room for longing
which is not pain, because it is conscious of tender relations with

God that guarantee its fulfilment. "I am Thy servant." Therefore the
longing which has no words needs none. "Thou hast loosed my
bonds." His thoughts go back to "the cords of death" (ver. 3), which
had held him so tightly. God's hand has slackened them, and, by
freeing him from that bondage, has bound him more closely than
before to Himself. "Being made free from sin, ye became the slaves
of righteousness." So, in the full blessedness of received deliverance,
the grateful heart offers itself to God, as moved by His mercies to
become a living sacrifice, and calls on the Name of Jehovah, in its
hour of thankful surrender, as it had called on that Name in its time
of deep distress. Once more the lonely suppliant, who had waded
such deep waters without companion but Jehovah, seeks to feel
himself one of the glad multitude in the courts of the house of
Jehovah, and to blend his single voice in the shout of a nation's
praise. We suffer and struggle for the most part alone. Grief is a
hermit, but Joy is sociable; and thankfulness desires listeners to its
praise. The perfect song is the chorus of a great "multitude which no
man can number."

PSALM CXVII.
1 Praise Jehovah, all nations,
Laud Him, all peoples.
2 For great is His lovingkindness over us,
And the troth of Jehovah endures for ever.
Hallelujah.
This shortest of the psalms is not a fragment, though some MSS.
attach it to the preceding and some to the following psalm. It
contains large "riches in a narrow room," and its very brevity gives
force to it. Paul laid his finger on its special significance, when he
quoted it in proof that God meant His salvation to be for the whole
race. Jewish narrowness was an after-growth and a corruption. The
historical limitations of God's manifestation to a special nation were
means to its universal diffusion. The fire was gathered in a grate,
that it might warm the whole house. All men have a share in what
God does for Israel. His grace was intended to fructify through it to
all. The consciousness of being the special recipients of Jehovah's
mercy was saved from abuse, by being united with the
consciousness of being endowed with blessing that they might
diffuse blessing.
Nor is the psalmist's thought of what Israel's experience proclaimed
concerning God's character less noteworthy. As often, lovingkindness
is united with troth or faithfulness as twin stars which shine out in all
God's dealings with His people. That lovingkindness is "mighty over
us"—the word used for being mighty has the sense of prevailing,
and so "where sin abounded, grace did much more abound." The
permanence of the Divine Lovingkindness is guaranteed by God's
Troth, by which the fulfilment of every promise and the prolongation
of every mercy are sealed to men. These two fair messengers have
appeared in yet fairer form than the psalmist knew, and the world

has to praise Jehovah for a world-wide gift, first bestowed on and
rejected by a degenerate Israel, which thought that it owned the
inheritance, and so lost it.

PSALM CXVIII.
1 Give thanks to Jehovah, for He is good,
For His lovingkindness endures for ever.
2 O let Israel say,
That His lovingkindness endures for ever.
3 O let the house of Aaron say,
That His lovingkindness endures for ever.
4 O let those who fear Jah say,
That His lovingkindness endures for ever.
5 Out of the strait place I called on Jah,
Jah answered me [by bringing me out] into an open place.
6 Jehovah is for me, I will not fear,
What can man do to me?
7 Jehovah is for me, as my helper,
And I shall gaze on my haters.
8 Better is it to take refuge in Jehovah
Than to trust in man.
9 Better is it to take refuge in Jehovah
Than to trust in princes.
10 All nations beset me round about;
In the name of Jehovah will I cut them down.
11 They have beset me round about, yea, round about beset me;
In the name of Jehovah will I cut them down.
12 They beset me round about like bees,
They were extinguished like a thorn fire;
In the name of Jehovah will I cut them down.
13 Thou didst thrust sore at me that I might fall,
But Jehovah helped me.
14 Jah is my strength and song,

And He is become my salvation.
15 The sound of shrill shouts of joy and salvation is [heard] in the
tents of the righteous;
The right hand of Jehovah does prowess.
16 The right hand of Jehovah is exalted,
The right hand of Jehovah does prowess.
17 I shall not die, but live,
And I tell forth the works of Jah.
18 Jah has chastened me sore,
But to death He has not given me up.
19 Open ye to me the gates of righteousness,
I will go in by them, I will thank Jah.
20 This is the gate of Jehovah:
The righteous may go in by it.
21 I will thank Thee, for Thou hast answered me,
And art become my salvation.
22 The stone [which] the builders rejected
Is become the head [stone] of the corner.
23 From Jehovah did this come to pass,
It is wonderful in our eyes.
24 This is the day [which] Jehovah has made,
Let us leap for joy and be glad in it.
25 O, I beseech Thee, Jehovah, save, I beseech;
O, I beseech Thee, Jehovah, give prosperity.
26 Blessed be he that comes in the name of Jehovah,
We bless you from the house of Jehovah.
27 Jehovah is God, and He has given us light;
Order the bough-bearing procession,—
To the horns of the altar!
28 My God art Thou, and I will thank Thee,

My God, I will exalt Thee.
29 Give thanks to Jehovah, for He is good,
For His lovingkindness endures for ever.
This is unmistakably a psalm for use in the Temple worship, and
probably meant to be sung antiphonally, on some day of national
rejoicing (ver. 24). A general concurrence of opinion points to the
period of the Restoration from Babylon as its date, as in the case of
many psalms in this Book V., but different events connected with
that restoration have been selected. The psalm implies the
completion of the Temple, and therefore shuts out any point prior to
that. Delitzsch fixes on the dedication of the Temple as the occasion;
but the view is still more probable which supposes that it was sung
on the great celebration of the Feast of Tabernacles, recorded in
Neh. viii. 14-18. In later times ver. 25 was the festal cry raised while
the altar of burnt-offering was solemnly compassed, once on each of
the first six days of the Feast of Tabernacles, and seven times on the
seventh. This seventh day was called the "Great Hosanna; and not
only the prayers at the Feast of Tabernacles, but even the branches
of osiers (including the myrtles), which are bound to the palm
branch (Lulab), were called Hosannas" (Delitzsch). The allusions in
the psalm fit the circumstances of the time in question. Stier,
Perowne, and Baethgen concur in preferring this date: the last-
named critic, who is very slow to recognise indications of specific
dates, speaks with unwonted decisiveness, when he writes, "I
believe that I can say with certainty, Psalm cxviii. was sung for the
first time at the Feast of Tabernacles in the year 444 b.c." Cheyne
follows his usual guides in pointing to the purification and
reconstruction of the Temple by Judas Maccabæus as "fully
adequate to explain alike the tone and the expressions." He is "the
terrible hero," to whose character the refrain, "In the name of
Jehovah I will cut them down," corresponds. But the allusions in the
psalm are quite as appropriate to any other times of national
jubilation and yet of danger, such as that of the Restoration, and

Judas the Maccabee had no monopoly of the warrior trust which
flames in that refrain.
Apparently the psalm falls into two halves, of which the former (vv.
1-16) seems to have been sung as a processional hymn while
approaching the sanctuary, and the latter (vv. 17-29), partly at the
Temple gates, partly by a chorus of priests within, and partly by the
procession when it had entered. Every reader recognises traces of
antiphonal singing; but it is difficult to separate the parts with
certainty. A clue may possibly be found by noting that verses marked
by the occurrence of "I," "me," and "my" are mingled with others
more impersonal. The personified nation is clearly the speaker of the
former class of verses, which tells a connected story of distress,
deliverance, and grateful triumph; while the other less personal
verses generalise the experience of the first speaker, and sustain
substantially the part of the chorus in a Greek play. In the first part
of the psalm we may suppose that a part of the procession sang the
one and another portion the other series; while in the second part
(vv. 17-29) the more personal verses were sung by the whole
cortège arrived at the Temple, and the more generalised other part
was taken by a chorus of priests or Levites within the sanctuary. This
distribution of verses is occasionally uncertain, but on the whole is
clear, and aids the understanding of the psalm.
First rings out from the full choir the summons to praise, which
peculiarly belonged to the period of the Restoration (Ezra iii. 11;
Psalms cvi. 1, cvii. 1). As in Psalm cxv., three classes are called on:
the whole house of Israel, the priests, and "those who fear
Jehovah"—i.e., aliens who have taken refuge beneath the wings of
Israel's God. The threefold designation expresses the thrill of joy in
the recovery of national life; the high estimate of the priesthood as
the only remaining God-appointed order, now that the monarchy was
swept away; and the growing desire to draw the nations into the
community of God's people.
Then, with ver. 5, the single voice begins. His experience, now to be
told, is the reason for the praise called for in the previous verses. It

is the familiar sequence reiterated in many a psalm and many a life,
—distress, or "a strait place" (Psalm cxvi. 3), a cry to Jehovah, His
answer by enlargement, and a consequent triumphant confidence,
which has warrant in the past for believing that no hand can hurt
him whom Jehovah's hand helps. Many a man passes through the
psalmist's experience without thereby achieving the psalmist's
settled faith and power to despise threatening calamities. We fail
both in recounting clearly to ourselves our deliverances and in
drawing assurance from them for the future. Ver. 5b is a pregnant
construction. He "answered me in [or, into] an open place"—i.e., by
bringing me into it. The contrast of a narrow gorge and a wide plain
picturesquely expresses past restraints and present freedom of
movement. Ver. 6 is taken from Psalm lvi. 9, 11; and ver. 7 is
influenced by Psalm liv. 4, and reproduces the peculiar expression
occurring there, "Jehovah is among my helpers,"—on which compare
remarks on that passage.
Vv. 8, 9, are impersonal, and generalise the experience of the
preceding verses. They ring out loud, like a trumpet, and are the
more intense for reiteration. Israel was but a feeble handful. Its very
existence seemed to depend on the caprice of the protecting kings
who had permitted its return. It had had bitter experience of the
unreliableness of a monarch's whim. Now, with superb reliance,
which was felt by the psalmist to be the true lesson of the
immediate past, it peals out its choral confidence in Jehovah with a
"heroism of faith which may well put us to the blush." These verses
surpass the preceding in that they avow that faith in Jehovah makes
men independent of human helpers, while the former verses
declared that it makes superior to mortal foes. Fear of and
confidence in man are both removed by trust in God. But it is
perhaps harder to be weaned from the confidence than to rise above
the fear.
The individual experience is resumed in vv. 10-14. The energetic
reduplications strengthen the impression of multiplied attacks,
corresponding with the facts of the Restoration period. The same

impression is accentuated by the use in ver. 11a of two forms of the
same verb, and in ver. 12a by the metaphor of a swarm of angry
bees (Deut. i. 44). Numerous, venomous, swift, and hard to strike at
as the enemies were, buzzing and stinging around, they were but
insects after all, and a strong hand could crush them. The psalmist
does not merely look to God to interpose for him, as in vv. 6, 7, but
expects that God will give him power to conquer by the use of his
own strengthened arm. We are not only objects of Divine protection,
but organs of Divine power. Trusting in the revealed character of
Jehovah, we shall find conquering energy flowing into us from Him,
and the most fierce assaults will die out as quickly as a fire of dry
thorn twigs, which sinks into ashes the sooner the more it crackles
and blazes. Then the psalmist individualises the multitude of foes,
just as the collective Israel is individualised, and brings assailants
and assailed down to two antagonists, engaged in desperate duel.
But a third Person intervenes. "Jehovah helped me" (ver. 13); as in
old legends, the gods on their immortal steeds charged at the head
of the hosts of their worshippers. Thus delivered, the singer breaks
into the ancient strain, which had gone up on the shores of the
sullen sea that rolled over Pharaoh's army, and is still true after
centuries have intervened: "Jah is my strength and song, and He is
become my salvation." Miriam sang it, the restored exiles sang it,
tried and trustful men in every age have sung and will sing it, till
there are no more foes; and then, by the shores of the sea of glass
mingled with fire, the calm victors will lift again the undying "song of
Moses and of the Lamb."
Vv. 15, 16, are probably best taken as sung by the chorus,
generalising and giving voice to the emotions excited by the
preceding verses. The same reiteration which characterised vv. 8, 9,
reappears here. Two broad truths are built on the individual voice's
autobiography: namely, that trust in Jehovah and consequent
conformity to His law are never in vain, but always issue in joy; and
that God's power, when put forth, always conquers. "The tents of
the righteous" may possibly allude to the "tabernacles" constructed
for the feast, at which the song was probably sung.

Vv. 17-19 belong to the individual voice. The procession has reached
the Temple. Deeper thoughts than before now mark the retrospect
of past trial and deliverance. Both are recognised to be from
Jehovah. It is He who has corrected, severely indeed, but still "in
measure, not to bring to nothing, but to make capable and recipient
of fuller life." The enemy thrust sore, with intent to make Israel fall;
but God's strokes are meant to make us stand the firmer. It is
beautiful that all thought of human foes has faded away, and God
only is seen in all the sorrow. But His chastisement has wider
purposes than individual blessedness. It is intended to make its
objects the heralds of His name to the world. Israel is beginning to
lay to heart more earnestly its world-wide vocation to "tell forth the
works of Jehovah." The imperative obligation of all who have
received delivering help from Him is to become missionaries of His
name. The reed is cut and pared thin and bored with hot irons, and
the very pith of it extracted, that it may be fit to be put to the
owner's lips, and give out music from his breath. Thus conscious of
its vocation and eager to render its due of sacrifice and praise, Israel
asks that "the gates of righteousness" may be opened for the
entrance of the long procession. The Temple doors are so called,
because Righteousness is the condition of entrance (Isa. xxvi. 2:
compare Psalm xxiv.).
Ver. 20 may belong to the individual voice, but is perhaps better
taken as the answer from within the Temple, of the priests or Levites
who guarded the closed doors, and who now proclaim what must be
the character of those who would tread the sacred courts. The gate
(not as in ver. 19, gates) belongs to Jehovah, and therefore access
by it is permitted to none but the righteous. That is an everlasting
truth. It is possible to translate, "This is the gate to Jehovah"—i.e.,
by which one comes to His presence; and that rendering would bring
out still more emphatically the necessity of the condition laid down:
"Without holiness no man shall see the Lord."
The condition is supposed to be met; for in ver. 21 the individual
voice again breaks into thanksgiving, for being allowed once more to

stand in the house of Jehovah. "Thou hast answered me": the
psalmist had already sung that Jah had answered him (ver. 5). "And
art become my salvation": he had already hailed Jehovah as having
become such (ver. 14). God's deliverance is not complete till full
communion with Him is enjoyed. Dwelling in His house is the crown
of all His blessings. We are set free from enemies, from sins and
fears and struggles, that we may abide for ever with Him, and only
then do we realise the full sweetness of His redeeming hand, when
we stand in His presence and commune evermore with Him.
Vv. 22, 23, 24, probably belong to the priestly chorus. They set forth
the great truth made manifest by restored Israel's presence in the
rebuilt Temple. The metaphor is suggested by the incidents
connected with the rebuilding. The "stone" is obviously Israel, weak,
contemptible, but now once more laid as the very foundation stone
of God's house in the world. The broad truth taught by its history is
that God lays as the basis of His building—i.e., uses for the
execution of His purposes—that which the wisdom of man despises
and tosses aside. There had been abundant faint-heartedness
among even the restored exiles. The nations around had scoffed at
these "feeble Jews," and the scoffs had not been without echoes in
Israel itself. Chiefly, the men of position and influence, who ought to
have strengthened drooping courage, had been infected with the
tendency to rate low the nation's power, and to think that their
enterprise was destined to disaster. But now the Temple is built, and
the worshippers stand in it. What does that teach but that all has
been God's doing? So wonderful is it, so far beyond expectation, that
the very objects of such marvellous intervention are amazed to find
themselves where they stand. So rooted is our tendency to unbelief
that, when God does what He has sworn to do, we are apt to be
astonished with a wonder which reveals the greatness of our past
incredulity. No man who trusts God ought to be surprised at God's
answers to trust.
The general truth contained here is that of Paul's great saying, "God
hath chosen the weak things of the world that He might put to

shame the things that are strong." It is the constant law, not
because God chooses unfit instruments, but because the world's
estimates of fitness are false, and the qualities which it admires are
irrelevant with regard to His designs, while the requisite qualities are
of another sort altogether. Therefore, it is a law which finds its
highest exemplification in the foundation for God's true temple,
other than which can no man lay. "Israel is not only a figure of Christ
—there is an organic unity between Him and them. Whatever,
therefore, is true of Israel in a lower sense is true in its highest
sense of Christ. If Israel is the rejected stone made the head of the
corner, this is far truer of Him who was indeed rejected of men, but
chosen of God and precious, the corner stone of the one great living
temple of the redeemed" (Perowne).
Ver. 24 is best regarded as the continuation of the choral praise in
vv. 22, 23. "The day" is that of the festival now in process, the joyful
culmination of God's manifold deliverances. It is a day in which joy is
duty, and no heart has a right to be too heavy to leap for gladness.
Private sorrows enough many of the jubilant worshippers no doubt
had, but the sight of the Stone laid as the head of the corner should
bring joy even to such. If sadness was ingratitude and almost
treason then, what sorrow should now be so dense that it cannot be
pierced by the Light which lighteth every man? The joy of the Lord
should float, like oil on stormy waves, above our troublous sorrows,
and smooth their tossing.
Again the single voice rises, but not now in thanksgiving, as might
have been expected, but in plaintive tones of earnest imploring (ver.
25). Standing in the sanctuary, Israel is conscious of its perils, its
need, its weakness, and so with pathetic reiteration of the particle of
entreaty, which occurs twice in each clause of the verse, cries for
continued deliverance from continuing evils, and for prosperity in the
course opening before it. The "day" in which unmingled gladness
inspires our songs has not yet dawned, fair as are the many days
which Jehovah has made. In the earthly house of the Lord
thanksgiving must ever pass into petition. An unending day comes,

when there will be nothing to dread, and no need for the sadder
notes occasioned by felt weakness and feared foes.
Vv. 26, 27, come from the chorus of priests, who welcome the
entering procession, and solemnly pronounce on them the
benediction of Jehovah. They answer, in His name, the prayer of ver.
25, and bless the single leader of the procession and the multitudes
following. The use of ver. 26a and of the "Hosanna" (an attempted
transliteration of the Hebrew "Save I beseech") from ver. 25 at
Christ's entrance into Jerusalem probably shows that the psalm was
regarded as Messianic. It is so, in virtue of the relation already
referred to between Israel and Christ. He "cometh in the name of
Jehovah" in a deeper sense than did Israel, the servant of the Lord.
Ver. 27a recalls the priestly benediction (Numb. vi. 25), and
thankfully recognises its ample fulfilment in Israel's history, and
especially in the dawning of new prosperity now. Ver. 27b, c, is
difficult. Obviously it should be a summons to worship, as
thanksgiving for the benefits acknowledged in a. But what is the act
of worship intended is hard to say. The rendering "Bind the sacrifice
with cords, even unto the horns of the altar," has against it the usual
meaning of the word rendered sacrifice, which is rather festival, and
the fact that the last words of the verse cannot possibly be
translated "to the horns," etc., but must mean "as far as" or "even
up to the horns," etc. There must therefore be a good deal supplied
in the sentence; and commentators differ as to how to fill the gap.
Delitzsch supposes that "the number of the sacrificial animals is to
be so great that the whole space of the courts of the priests
becomes full of them, and the binding of them has therefore to take
place even up to the horns of the altar." Perowne takes the
expression to be a pregnant one for "till [the victim] is sacrificed and
its blood sprinkled on the horns of the altar." So Hupfeld, following
Chaldee and some Jewish interpreters. Others regard the supposed
ellipsis as too great to be natural, and take an entirely different view.
The word rendered sacrifice in the former explanation is taken to
mean a procession round the altar, which is etymologically

justifiable, and is supported by the known custom of making such a
circuit during the Feast of Tabernacles. For "cords" this explanation
would read branches or boughs, which is also warranted. But what
does "binding a procession with boughs" mean? Various answers are
given. Cheyne supposes that the branches borne in the hands of the
members of the procession were in some unknown way used to bind
or link them together before they left the Temple. Baethgen takes
"with boughs" as = "bearing boughs," with which he supposes that
the bearers touched the altar horns, for the purpose of transferring
to themselves the holiness concentrated there. Either explanation
has difficulties,—the former in requiring an unusual sense for the
word rendered sacrifice; the latter in finding a suitable meaning for
that translated bind. In either c is but loosely connected with b, and
is best understood as an exclamation. The verb rendered bind is
used in 1 Kings xx. 14, 2 Chron. xiii. 3, in a sense which fits well
with "procession" here—i.e., that of marshalling an army for battle.
If this meaning is adopted, b will be the summons to order the
bough-bearing procession, and c a call to march onwards, so as to
encircle the altar. This meaning of the obscure verse may be
provisionally accepted, while owning that our ignorance of the
ceremonial referred to prevents complete understanding of the
words.
Once more Miriam's song supplies ancient language of praise for
recent mercies, and the personified Israel compasses the altar with
thanksgiving (ver. 28). Then the whole multitude, both of those who
had come up to the Temple and of those who had welcomed them
there, join in the chorus of praise with which the psalm begins and
ends, and which was so often pealed forth in those days of early joy
for the new manifestations of that Lovingkindness which endures
through all days, both those of past evil and those of future hoped-
for good.

PSALM CXIX.
It is lost labour to seek for close continuity or progress in this psalm.
One thought pervades it—the surpassing excellence of the Law; and
the beauty and power of the psalm lie in the unwearied reiteration of
that single idea. There is music in its monotony, which is subtilely
varied. Its verses are like the ripples on a sunny sea, alike and
impressive in their continual march, and yet each catching the light
with a difference, and breaking on the shore in a tone of its own. A
few elements are combined into these hundred and seventy-six
gnomic sentences. One or other of the usual synonyms for the Law
—viz., word, saying, statutes, commandments, testimonies,
judgments—occurs in every verse, except vv. 122 and 132. The
prayers "Teach me, revive me, preserve me—according to Thy
word," and the vows "I will keep, observe, meditate on, delight in—-
Thy law," are frequently repeated. There are but few pieces in the
psalmist's kaleidoscope, but they fall into many shapes of beauty;
and though all his sentences are moulded after the same general
plan, the variety within such narrow limits is equally a witness of
poetic power which turns the fetters of the acrostic structure into
helps, and of devout heartfelt love for the Law of Jehovah.
The psalm is probably of late date; but its allusions to the singer's
circumstances, whether they are taken as autobiographical or as
having reference to the nation, are too vague to be used as clues to
the period of its composition. An early poet is not likely to have
adopted such an elaborate acrostic plan, and the praises of the Law
naturally suggest a time when it was familiar in an approximately
complete form. It may be that the rulers referred to in vv. 23, 46,
were foreigners, but the expression is too general to draw a
conclusion from. It may be that the double-minded (ver. 113), who
err from God's statutes (ver. 118), and forsake His law (ver. 53), are
Israelites who have yielded to the temptations to apostatise, which

came with the early Greek period, to which Baethgen, Cheyne, and
others would assign the psalm. But these expressions, too, are of so
general a nature that they do not give clear testimony of date.
§ א

1 Blessed the perfect in [their] way,
Who walk in the law of Jehovah!
2 Blessed they who keep His testimonies,
That seek Him with the whole heart,
3 [Who] also have done no iniquity,
[But] have walked in His ways!
4 Thou hast commanded Thy precepts,
That we should observe them diligently.
5 O that my ways were established
To observe Thy statutes!
6 Then shall I not be ashamed,
When I give heed to all Thy commandments.
7 I will thank Thee with uprightness of heart,
When I learn Thy righteous judgments.
8 Thy statutes will I observe;
Forsake me not utterly.
The first three verses are closely connected. They set forth in
general terms the elements of the blessedness of the doers of the
Law. To walk in it—i.e., to order the active life in conformity with its
requirements—ensures perfectness. To keep God's testimonies is at
once the consequence and the proof of seeking Him with whole-
hearted devotion and determination. To walk in His ways is the
preservative from evil-doing. And such men cannot but be blessed
with a deep sacred blessedness, which puts to shame coarse and
turbulent delights, and feeds its pure fires from God Himself.
Whether these verses are taken as exclamation or declaration, they
lead up naturally to ver. 4, which reverently gazes upon the loving
act of God in the revelation of His will in the Law, and bethinks itself
of the obligations bound on us by that act. It is of God's mercy that
He has commanded, and His words are meant to sway our wills,
since He has broken the awful silence, not merely to instruct us, but
to command; and nothing short of practical obedience will discharge
our duties to His revelation. So the psalmist betakes himself to
prayer, that he may be helped to realise the purpose of God in giving

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Advances In Animal Genomics Sukanta Mondal Editor Ram Lakhan Singh

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