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Animal Research

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Title: Animal Research


1
Animal Research In the Genomics Era
Martina McGloughlin, Biotechnology Program and
Life Sciences Informatics Program UC Davis
2
One Human Sequence
Typed in 10-pitch font, one human sequence would
stretch for more than 5,000 miles. Digitally
formatted, it could be stored on one CD-ROM.
Biologically encoded, it fits easily within a
single cell.
3
Paradigm Shift in Biology
The new paradigm, now emerging, is that all the
genes will be known (in the sense of being
resident in databases available electronically),
and that the starting point of a biological
investigation will be theoretical. An individual
scientist will begin with a theoretical
conjecture, only then turning to experiment to
follow or test that hypothesis.
Walter Gilbert. 1991. Towards a paradigm shift
in biology. Nature, 34999.
4
Paradigm Shift in Biology
To use the flood of knowledge, which will pour
across the computer networks of the world,
biologists not only must become computer
literate, but also change their approach to the
problem of understanding life.
Walter Gilbert. 1991. Towards a paradigm shift
in biology. Nature, 34999.
5
Animal Biotechnology- Applications
  • Recombinant vaccines and therapeutics, e.g
    rabies, rinderpest in vaccinia delivery
    vectors
  • Marker assisted selection
  • Supplementals, recombinant Bovine Somatatropin
  • Transgenics
  • Agriculture Applications
  • - disease resistance
  • - improved productivity
  • improved growth rate
  • improved metabolism
  • improved milk quality
  • improved meat quality
  • reduced fat
  • Medical Applications
  • - to produce valuable proteins in milk, blood
    or urine
  • - Xenotransplantation

6
Animal Biotechnology- Technologies
  • Technology Application
  • Antisense, ribozymes, To turn off or down
    undesirable
  • cosupression traits such as milk fat
    production, lactose
  • Embryonic Stem Cells Replace faulty tissues
  • Drug toxicology testing
  • (primordial germ cells) To Increase
    transformation efficiencies
  • Viral/Sperm delivery To Increase Transformation
    efficiencies
  • Nuclear Transplantation To improve transformation
    efficiencies and reproducibility
  • To allow herd trait consistency
  • Homologous Recombination Site specific gene
    insertion/ removal
  • Transient Expression To transiently transform
    mammary cells to study heterologous protein
    expression
  • Genomics, proteomics, To study global level gene
    expression
  • Technology Application
  • Antisense, ribozymes, To turn off or down
    undesirable
  • cosupression traits such as milk fat
    production, lactose
  • Genomics, proteomics, To study global level gene
    expression
  • bioinformatics To select, track and/or modify
    genes coding for valuable traits eg caseins
    for cheese
  • Embryonic Stem Cells Replace faulty tissues
  • Drug toxicology testing
  • (primordial germ cells) To Increase
    transformation efficiencies
  • Nuclear Transplantation To improve transformation
    efficiencies and reproducibility
  • To allow herd trait consistency
  • Transient Expression To transiently transform
    mammary cells to study heterologous protein
    expression
  • Homologous Recombination Site specific gene
    insertion/ removal

7
Genomics
  • Changes that will have effects comparable to
    those of the Industrial Revolution and the
    computer-based revolution are now beginning. The
    massive interest and commitment of resources in
    both the public and private sectors flows from
    the generally-held perception that genomics will
    be the single most fruitful approach to the
    acquisition of new information in basic and
    applied biology in the next several decades.
  • If genomics were only to be a tool for the basic
    biologist, the benefits of this approach would be
    staggering, yielding new insights into
    fundamental processes such as cell division,
    differentiation, transformation, the development
    and reproduction of organisms and the diversity
    of populations.
  • The rewards in applied biology, however, have
    clearly attracted the private sector and public
    interest. These include the promise of facile new
    approaches for drug discovery, new understanding
    of how cancers form and new approaches to
    determining qualitative and quantitative traits
    in animals for breeding and genetic engineering.

8
Human Genome Project - Goals
  • Construction of a high-resolution genetic map
  • Production of a variety of physical maps of all
    human chromosomes and of selected model organisms
  • Determination of the complete sequence of human
    DNA and DNA of selected model organisms
  • Development of capabilities for collecting,
    storing, distributing, and analyzing the data
    produced
  • Creation of appropriate technologies necessary to
    achieve these objectives

9
Sequencing Programs
Organism of genes genes with Comp. date
inferred function for genome
sequencing E. Coli 4,288 60 1997 Y
east 6,600 40 1996 C. Elegans
19,000 40 1998 Drosophila 12,000-14,000 25
1999 Arabidopsis 25,000 40 2000 Mouse
60,000-100,000 10-20 2002 Human
60,000-100,000 10-20 2000/3
10
The Shape of the Wave
  • 1999
  • JGI releases 150 Mbases draft
  • Celera releases the sequence of Drosophila (140
    Mb)
  • Public draft effort reaches halfway point
    (1,500 Mb)
  • 20 more Microbial genomes completed (80 Mb but
    60,000 genes)
  • First release of Celera shotgun (9,000 Mb)
  • 2000
  • Public draft completed (1,500 Mb)
  • Mouse draft begins (500 Mb - comparisons with
    human)
  • Two more Celera shotgun releases ( 18,000 Mb)
  • 40 more Microbial genomes sequenced (160 Mb
    -120,000 genes)

11
Genomics, Proteomics and Bioinformatics
  • Genomics is operationally defined as
    investigations into the structure and function of
    very large numbers of genes undertaken in a
    simultaneous fashion.
  • Structural genomics includes the genetic mapping,
    physical mapping and sequencing of entire
    genomes.
  • Comparative genomics means information gained in
    one organism can have application in other even
    distantly related organisms. This enables the
    application of information gained from facile
    model systems to agricultural and medical
    problems. The nature and significance of
    differences between genomes also provides a
    powerful tool for determining the relationship
    between genotype and phenotype through
    comparative genomics and morphological and
    physiological studies.
  • Functional genomics Phenotype is logically the
    subject of functional genomics. Genome sequencing
    for most organisms of interest will be complete
    within the near future, ushering in the so called
    "post-genome era."

12
Genomics, Proteomics and Bioinformatics
  • Proteomics At the molecular level, phenotype
    includes all temporal and spatial aspects of gene
    expression as well as related aspects of the
    expression, structure, function and spatial
    localization of proteins. The Proteome is the set
    of all expressed proteins for a given organism.
  • The next hierarchical level of phenotype
    considers how the proteome within and among cells
    cooperates to produce the biochemistry and
    physiology of individual cells and organisms.
    PhysiomicsMetanomics is a descriptor for this
    approach. Phenomics" The final hierarchical
    levels of phenotype include anatomy and function
    for cells and whole organisms.
  • Bioinformatics Computational or algorithmic
    approaches to the production of information from
    large amounts of biological data, include
    prediction of protein structure, dynamic modeling
    of complex physiological systems or the
    statistical treatment of quantitative traits in
    populations in order to determine the genetic
    basis for these traits.
  • Unquestionably, bioinformatics will be an
    essential component of all research activities
    utilizing structural and functional genomics
    approaches

13
Fundamental Dogma
Although many databases already exist to
distribute molecular information,
the post-genomic era will need many more to
collect, manage, and publish the coming flood of
new findings.
If this extension covers functional genomics,
then functional genomics is equivalent to
biology.
14
Structural genomics includes the genetic mapping,
physical mapping and sequencing of entire
genomes.
15
How to do Structural genomics physical mapping
and sequencing of entire genomes.
16
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17
Livestock Animal Genome Databases
Database Development Development of the livestock
animal genome database began in 1991 by Alan
Hillyard of the Jackson Laboratory, Bar Harbor,
Maine. It is modeled after the Mouse Genome
Database (MGD) and uses Ingres relational
database software. Each livestock species has a
separate database that is a collaboration among
many researchers and institutions. In 1994,
work began on the next version of the animal
genome database at the Roslin Institute in
Edinburgh, Scotland. The database was redesigned
around "experiments" in order to capture data
derived from new molecular genetic techniques.
National Animal Genome Research Program In the
United States, the National Animal Genome
Research Program (NAGRP) is responsible for
genome research and mapping of agriculturally
important livestock and aquatic species. The
NAGRP established four Species Genome Committees
in 1992 to coordinate the animal genome research
effort. Each species (bovine, poultry, sheep and
swine) has a Species Coordinator who facilitates
the genome research, mapping and database
activities.
18
Livestock Animal Genome Databases
  • Aggressive genome projects on agriculturally
    important animal species cattle, pig, sheep,
    horse, and chicken yielded powerful tools for
    assessing genes that specify hereditary
    disorders infectious disease resistance
    breed-specific quantitative trait loci (QTLs)
    phenotypes of agricultural relevance - economic
    trait loci (ETLs).
  • Genetic identification and tracking of ETLs in
    animal pedigrees have considerable import for
    livestock improvement and production. Early maps
    for farm animals emphasized STR markers, as they
    are informative in pedigrees segregating disease
    or ETLs.
  • Comparative anchor loci (CAL) provide
    cross-reference to the gene-rich mouse and human
    maps now critical.
  • CAL maps allow "comparative candidate positional
    cloning"a three-step gene identification strategy
    that
  • assesses the linkage of an animal variant
    phenotype to a specific chromosomal position
  • identifies responsible candidate genes in that
    region by inspecting the homologous region of the
    human and mouse gene maps using CAL as landmarks
    to demarcate chromosomal regions
  • identifies and genotypes SNP markers in or around
    the candidate loci - tests for association (or
    not) of these with the phenotype.

19
ANIMAL GENOME AND GENETIC MECHANISMS
  • USDA The objective of this program is to
    increase our knowledge and understanding of the
    structure, organization, function, regulation and
    expression of genes in agriculturally important
    animals including aquaculture species.
  • This includes but is not limited to
  • comparative gene mapping
  • the identification, isolation, characterization
    of genes, gene products and their regulatory
    mechanisms
  • identification and mapping of DNA segregation
    markers including quantitative trait loci (QTL)
  • interactions between nuclear and organellar genes
  • the molecular basis of genetic replication
  • development and application of methods to modify
    the animal genome.
  • A new animal genome basic reagents and tools
    section was introduced in 1999 to initiate large
    scale EST sequencing, arrayed BAC libraries, DNA
    microarrays and high resolution genetic maps.

20
Livestock Animal Genomics
  • Marker-Assisted Selection MAS
  • ID chromosomal regions for genes using a
    whole-genome-scanning approach has stimulated
    interest among cattle-breeding industry
    organizations.
  • Markers used to select for genetically superior
    bulls and heifers
  • Qualitative Traits Horn development -Weaver
    syndrome
  • Quantitative Traits Milk production and somatic
    cell counts
  • genotypes for kappa casein and beta lactoglobulin
  • pedigree, family, and individual performance
    statistics.
  • Disorders Enzyme, biological activity, or
    DNA-PCR-based tests are used to screen for
    autosomal recessive disorders such as bovine
    citrullinemia, protoporphyria, factor XI
    deficiency, bovine leukocyte adhesion deficiency,
    and uridine monophosphate synthase deficiency.
  • Parent ID Microsatellites now used for parentage
    identification in addition to or in place of
    blood groups.
  • OTLs discovery and characterization of the actual
    genes (QTLs) responsible for the phenotypic
    variation of a given trait.
  • Positional cloning of interesting genes

21
Marker-Assisted Selection MAS- QTLs
Most traits are not controlled by a single gene,
but rather by a set of genes acting in concert.
Traits that are controlled by multiple genes,
such as size, tend to have a more subtle,
constant variation in the result phenotype,
leading to a continuous distribution of
phenotypic values. These are also known as
"quantitative" traits. Traits controlled by one
or a very few genes, such as certain diseases,
tend to have more drastic, discrete changes in
expression, and these are termed "qualitative"
traits. Single gene traits- many genes mapped
to relatively precise locations by observing the
segregation of the trait in a mapping
population. Quantitative traits - too difficult
to distinguish changes in phenotype, so special
QTL-mapping studies must be done. Set of
intervals, not points, scattered over the genome,
in which a gene or genes affecting the trait is
thought to lie. Depending on how the study was
done, these intervals may be centered over a
single marker, or lie in between two markers.
The result of the study is a list of markers
whose particular alleles tend to be associated
with certain changes in the traits being
evaluated.

22
Marker-Assisted Selection MAS- Syntenic
relationshipss\
Synteny As more is learned about the content and
organization of the genomes of various organisms,
scientists are learning that "blocks" of genes
are often preserved in some fashion across
species. In other words, a segment of chromosome
3 in sheep may have the same genes, in the same
order, as a segment of chromosome 7 in goats.
The more closely related the species are, the
more likely it is that we can detect homologous
segments. Much of the currently available
information is based on a fairly gross scale (the
"blocks" may be many, many cM big) and we are
still learning more about how well these
relationships hold up at a finer scale. Knowing
where these homologous segments are can help you
leverage the information available from a
well-studied organism, or extrapolate information
from one species to another. For example, if you
know where an important gene is located in mice,
you might be able to predict where its
counterpart is located in pig.
23
Marker-Assisted Selection MAS- Techniques
Most genetic maps consist of anonymous molecular
markers and not genes of known function.
Molecular markers can be generated by a variety
of different techniques, each with their
advantages and disadvantages, and different sorts
of information will accompany the different
marker types. These markers may pinpoint genes,
(for example cDNA, RFLP or EST markers), or
proteins, (isozyme markers), or they may mark
genomic DNA (for example genomic RFLP, RAPD or
microsatellite markers). They may be gel-based,
or PCR-based they may detect a single locus or
multiple loci they may or may not require
sequence information finally, they may vary in
their degree of reliability, difficulty and
expense, and the nature of the polymorphism they
detect. A user needs to be somewhat familiar
with a particular technology to assess whether a
database is providing adequate and useful
information.
24
Marker-Assisted Selection MAS- Techniques
RFLPs Restriction Fragment Length Polymorphism.
This technique requires that you create a library
of DNA fragments cloned into some vector. These
probe libraries may be based on genomic or cDNA.
It does not require sequencing, and is gel-based,
not PCR-based. The DNA of the organism you are
interested in is digested with restriction
enzymes and probed with the library of cloned
DNA. Matches to the probe DNA are visualized
using radioactivity. The technique is slow and
requires a significant investment of time. This
method detects a single locus, and the fact that
it will consistently detect the same locus makes
this technology well-suited for mapping,
comparative mapping and QTL studies. Polymorphism
is generally detected as a difference in the
molecular weight (and thus the migration through
a gel) of the fragments of host DNA.
25
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26
Marker-Assisted Selection MAS- Techniques
RAPD-PCR Randomly amplified polymorphic PCR This
is a marker technique that requires no cloning or
sequencing, is PCR-based and may detect several
loci simultaneously. Short (about 10 base pair)
random primer sequences are used to amplify DNA,
and generally results in a presence/absence
polymorphism. This technique is easy, inexpensive
and fast, and because a single RAPD primer may
detect more than one loci, it is useful for
phylogenetic studies. The short primers, however,
may be easily affected by annealing conditions
and results may not always be consistently
reproducible. This marker technology is not
appropriate for use in comparative mapping.
SCAR Sequence Characterized Amplified Region.
More robust and not as random as RAPDs.
27
Marker-Assisted Selection MAS- Techniques
AFLP Amplified Fragment length Polymorhisms
AFLPs are a PCR-based marker system involving
amplification of DNA after cleavage with
restriction enzymes. This technique also requires
no cloning or sequencing and is PCR-based. It
operates on much the same principle as a RAPD,
but the primer consists of a longer ( 15 bp)
fixed portion and a short (2-4 bp) random
portion. The long fixed portion gives the primer
stability and the short random portion means it
will amplify many loci - one may get over 100
loci amplified with a single AFLP primer.
Polymorphism is detected as the presence/absence
of a band. The amplification is not completely
random (as in the case of RAPDs) nor is it based
on a known host DNA sequence (as in the case of
SSRs). Rather, it is based on selective
amplification of subsets of tagged fragments.
Because it detects so many loci, it is very
useful for fingerprinting. Direct In-situ Single
Copy PCR (DISC-PCR) was developed to map short
unique sequences (even microsatellites) to
physical locations on chromosomes
28
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29
Marker-Assisted Selection MAS- Techniques
STS stands for Sequence Tagged Site. This is a
PCR-based approach that detects a single, unique,
sequence-defined point in the genome. This does
not require cloning, but does require sequence
information. Primers of 18-20 base pairs are
designed to amplify some short, unique fragment
of DNA whose sequence is known (for example, this
might be the sequence of a RAPD PCR product, or
an RFLP clone). Polymorphism is generally
detected as a size difference in the amplified
product if there is no size difference,
restriction enzymes may be used to cut the
products and identify polymorphism. Since the
primers are longer than RAPD primers and based on
a specific sequence, this method reliably detects
the same locus and is good for mapping studies.
The design and creation of good primers may
involve a significant investment.
30
Marker-Assisted Selection MAS- Techniques
EST stands for Expressed Sequence Tag. As part
of many sequencing projects, partial (end)
sequences of cDNA clones are generated. These
partial sequences can then be used to design
18-20 base pair primers which provide a unique
sequence tag for the gene. Again, this is a
PCR-based approach which does not require
cloning, but does require sequence information.
It detects a unique, expressed region of the
genome, and is good for mapping. Polymorphism is
generally detected as a size difference of the
amplified product. The design and creation of
good primers may involve a significant
investment.
31
Marker-Assisted Selection MAS- Techniques
Microsatellite markers detect hypervariable
regions of the genome. A microsatellite (also
called simple sequence repeat (SSR), short tandem
repeat (STR) or variable number tandem repeat
(VNTR)) is a short (2-5 base) motif that is
repeated multiple times and is flanked by unique
DNA. The microsatellite motif is used as a probe
against genomic or cDNA libraries to identify
clones containing the motif. These clones are
then end sequenced, and primers are designed to
amplify the unique DNA flanking the
microsatellite motif. This method is repeatable,
identifies a single locus, and targets
hypervariable regions of the genome. Polymorphism
is generally detected as a length difference in
the amplified product. This length difference may
be very small, for example 2 base pairs. It does
require a significant investment of time and
resources, and is appropriate for mapping, QTLs
studies and fingerprinting.
32
Marker-Assisted Selection MAS- Techniques
Single-nucleotide polymorphisms (SNPs) markers
are common bi-allelic within coding regions, or
more often in noncoding intron or intergenic
regions. SNPs are also valuable for pedigree,
family, or population screens within species,
particularly with automated array-based
genotyping technologies, but are usually
uninformative when used for comparative ortholog
identification between orders. Type III SNP
markers occur once every 500 to 1000 base pairs
(bp) in the human genome, totaling an estimated
3 million SNPs in the genomes of human and other
mammals of comparable within-species genetic
diversity. Zoo-FISH The application of
interspecies chromosome painting), whereby DNA
from fluorescent-labeled flow-sorted individual
chromosomes of one species is hybridized in situ
to metaphase spreads of a compared species, has
allowed identification of evolutionarily
conserved chromosomes, chromosome arms, and
segments virtually by direct observation
Zoo-FISH The application of interspecies
chromosome painting), whereby DNA from
fluorescent-labeled flow-sorted individual
chromosomes of one species is hybridized in situ
to metaphase spreads of a compared species, has
allowed identification of evolutionarily
conserved chromosomes, chromosome arms, and
segments virtually by direct observation
33
Marker-Assisted Selection MAS- techniques
  • Diagnostics
  • Genotyping polymorphisms, SNPs
  • Microarray
  • Celera, Incyte, Affymetrix, Molecular Dynamics
  • The SNP Consortium is an innovative collaboration
    among industry, medical research charity, and
    several academic institutions -- is organized as
    a non-profit entity whose goal is to create and
    make publicly available a high-quality SNP map of
    the human genome. (Customized Medicine)
  • Sequences
  • Expressed sequence tags (ESTs)
  • Micoarrays
  • Human Genome Science, Millennium, Celera, Incyte
  • Curagen, Genaissance, LION, Genzyme,
  • Molecular Dynamics and Affymetrix Genetic
    Analysis Technology Consortium (GATC)
    standardize the rapidly growing field of
    array-based genetic analysis, paving the way for
    the more affordable and productive development of
    therapeutic, diagnostic and disease management
    products.

34
Marker-Assisted Selection MAS- Problems
  • Statistical analysis procedures tend to
    overestimate the size of the effect contributed
    from a marker-flanked interval on a phenotype.
  • Due to
  • differences in number of alleles at flanking
    microsatellite site
  • combined with genetic noise coming from the
    mammalian genome
  • recombination differences among individuals,
  • epistatic gene effects,
  • genetic heterogeneity,
  • incomplete penetrance,
  • phenocopy,
  • mitochondrial inheritance, and
  • genomic imprinting

35
Marker-Assisted Selection MAS- Maximizing
  • The key components for maximizing genetic
    improvement for various market situations and
    traits depend on
  • Selection intensity- how well animals are
    evaluated or the accuracy of prediction
  • Standard deviation of additive genetic values -
    magnitude of genetic differences among animals
  • Generation interval how fast the genetically
    superior younger animals replace their parents.
  • The goal is to optimally make genetic progress in
    a market that is simultaneously driven by several
    selection objectives.

36
Marker-Assisted Selection MAS- Maximizing
  • CATTLE
  • Benefits from use of markers flanking QTLs are
  • Improvement in the graduation rate of bulls from
    progeny test programs and reduction for bulls in
    waiting at the A.I. facility.
  • As confidence in marker use builds, accuracy of
    prediction in the equation for genetic change
    would also increase.
  • The combined use of MAS with reproductive
    technologies to reduce generation interval, the
    most limiting variable in the equation for
    genetic gain for cattle, may be greatest benefit.
  • MAS and the recovery of fetal oocytes followed by
    in vitro maturation, fertilization, and transfer
    of the favored allele-bearing embryo to a
    surrogate dam. The prospect of rapid genetic
    improvement, like animals with shorter generation
    intervals, has immediate appeal to cattle
    breeders. Recovery of oocytes and subsequent in
    vitro maturation, fertilization, and transfer of
    embryos has been accomplished from 7 month
    heifers.
  • Sexing embryos and determining disease-allele
    inheritance status using whole genome
    amplification techniques for single cells and PCR
    primers targeted at a specific DNA sequence has
    become a routine procedure in research labs.

37
ANIMAL GENOME AND GENETIC MECHANISMS
Functional Genomics in Cattle To address the
present deficiencies in resources for cattle
genomics, we propose to generate 17,500 3'
expressed sequence tags (ESTs) from cattle cDNA
libraries and to map 700 new ESTs on a bovine
radiation hybrid (RH) panel. Results of this
project will provide essential tools for
identification of genes controlling traits of
economic importance to the U. S. dairy and beef
industries. Anticipate that this project will
result in identification and mapping of
approximately 7 of all cattle genes. These
genes will be a rich source of transcripts
involved in reproduction, development, growth,
metabolism and disease resistance. Mapped ESTs
will be used for building detailed comparative
maps, understanding genome evolution and leading
the way towards molecular identification of genes
affecting economically important traits.
Lewin, H.A. University of Illinois
Genomics in Cattle The muscular hypertrophy
(double muscling) trait was mapped with type II
markers to bovine chromosome 2. Comparative
candidate positional cloning suggested myostatin
as a candidate gene to Grobet et al., who
identified an 11-bp deletion responsible for the
trait in Belgian Blue cattle. To address the
present deficiencies in resources for cattle
genomics, Levin, UI proposes to generate 17,500
3' expressed sequence tags (ESTs) from cattle
cDNA libraries and to map 700 new ESTs on a
bovine radiation hybrid (RH) panel. Results of
this project will provide essential tools for
identification of genes controlling traits of
economic importance to the U. S. dairy and beef
industries. Anticipate that this project will
result in identification and mapping of
approximately 7 of all cattle genes. These genes
will be a rich source of transcripts involved in
reproduction, development, growth, metabolism and
disease resistance. Mapped ESTs will be used
for building detailed comparative maps,
understanding genome evolution and molecular
identification of genes affecting economically
important traits.
38
Marker-Assisted Selection MAS- Maximizing
SHEEP The randomly amplified polymorphic
polymerase chain reaction (RAPD-PCR) methodology
was optimized to analyze sheep DNA (Juan Medrano,
Animal Science). They used bulked segregant
analysis for analyzing specific chromosomal
regions, including the fecundity gene in
Boorooola sheep and the high- growth locus in
mice. Ovine yeast artificial chromosome (YAC)
library (Noelle Cockett). Cockett's laboratory
has demonstrated that the callipyge gene, which
is responsible for muscle hypertrophy in sheep,
maps to the telomeric end of ovine chromosome
21. Spider Lamb syndrome, or ovine hereditary
chondrodysplasia, is recessive trait common to
several breeds. Mapped to the distal end of ovine
chs 6, comparative map inspection showed that
homologous segments on human chs 4p16.3 and mouse
chrs 5.15 included the fibroblast growth factor
receptor 3 (FGFR3) locus. Human mutations and
mouse knockouts of FGFR3 showed skeletal
deformities similar to Spider Lamb syndrome.
Search 1000 affected sheep implicated causative
mutation in the ovine FGFR3 gene.
SHEEP The randomly amplified polymorphic
polymerase chain reaction (RAPD-PCR) methodology
was optimized to analyze sheep DNA (Juan Medrano,
Animal Science) They used bulked segregant
analysis for analyzing specific chromosomal
regions, including the fecundity gene in
Boorooola sheep and the high- growth locus in
mice. Ovine yeast artificial chromosome (YAC)
library (Noelle Cockett). Cockett's laboratory
has demonstrated that the callipyge gene, which
is responsible for muscle hypertrophy in sheep,
maps to the telomeric end of ovine chromosome 21.

39
Marker-Assisted Selection MAS- Maximizing
PIGS The first practical manipulation of a
major gene in pig breeding using molecular
biology tools was the case of the so-called
halothane or stress gene. Single point mutation
in the calcium release channel ryanodine receptor
gene (RYR1) in recessive condition is responsible
for porcine stress syndrome (PSS, malignant
hyperthermia) and also results in, or is closely
linked to, a gene or genes involved in
determining leanness. The detection of this
mutation using a polymerase chain
reaction-restriction fragment length polymorphism
(PCR-RFLP) test provides pig breeders with the
means to precisely control the distribution of
the mutation. As PSS is a recessive condition,
but effects on leanness are essentially additive,
elimination of the gene from parent females can
allow the use of high-lean-yield boars homozygous
for the mutation to boost the performance of
nonsusceptible (heterozygous) slaughter progeny.
40
Marker-Assisted Selection MAS- Maximizing
The PSS effect of the halothane gene is, however,
qualitative. What about genes that have major
effects on quantitative traits? The discovery
of highly polymorphic microsatellite sequences
has enabled the rapid development of genetic
linkage maps. These markers are now used to
search for loci affecting quantitative traits of
economic importance in the pig. Economic trait
loci (ETL) have been identified for lean growth
and intramuscular fat using divergent crosses
between European meat breeds and the Chinese
Meishan or European wild boar. The first use of
such markers is for introduction of desired genes
from exotic lines while minimizing the effect of
genes unfavorable for lean growth. Using
marker-assisted selection, the efficiency of
incorporating high-prolificacy genes from the
Chinese Meishan into meat breeds.
41
ANIMAL GENOME AND GENETIC MECHANISMS
Functional Genomics in the Pig An improved
understanding of porcine reproductive biology is
of crucial economic importance, yet reproductive
processes are poorly characterized at the
molecular level. Further, reproduction is very
difficult to improve by classical breeding
methods. The basic need to identify thousands
of genes associated with physiology of
reproduction and to create the necessary
infrastructure to share that information with
other scientists. The Specific Aims of their
research are to sequence a large number (20,000)
of genes from porcine reproductive tissues,
develop databases for public access, and map a
significant number of these genes (700) which
have been mapped in other species. All resources
produced from this process (cloned DNA for each
gene)will be publicly available and all data
(sequence and mapping) will be accessible through
an improved bioinformatics pipeline designed to
allow interested scientists to make use of the
information discovered. Tuggle, C.K. Iowa
State University
42
ANIMAL GENOME AND GENETIC MECHANISMS
Chicken The chicken genome is made up of 40
chromosomes which, in total, contain about 1.2
billion base pairs. Recombinant DNA technology
allows for the manipulation in BAC of chicken DNA
segments of 100-200,000 base pairs each, or about
200-400 segments per average chromosome. The goal
is to generate approximately 60,000 such
overlapping segments and determine physical map.
Use library to locate BAC containing the gene
encoding trait of interest. In this way, the
physiological basis for inherited production
traits will be better understood. That
understanding can then be applied to improved
management, nutrition and breeding strategies for
egg and meat productivity. - An Integrated BAC
Map of the Chicken Genome Dodgson, J.B,
MSU Using molecular markers in genetic tests,
elite progeny can be identified earlier and more
accurately. Cheng, H.H. Soller, M. USDA ARS have
identified in experimental chickens a number of
genes that promote resistance to Marek's disease,
the leading chronic disease of chickens and a 1
billion per year problem. The goal is to transfer
this information to commercial lines. They wish
to determine if their genetic tests accurately
predict disease resistance in elite flocks.
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Marker-Assisted Selection MAS- The future
The real advantage of marker-assisted selection
will be in nontraditional improvement schemes.
For example, improvements may include
preselection prior to conventional performance
testing to increase accuracy and selection
intensity and to identify traits that are sex- or
age-limited or difficult to test or measure, such
as meat quality and disease susceptibility. In
the near future, many additional markers
associated with QTL will be discovered and the
capability to screen cheaply and rapidly for many
DNA markers will be available, allowing breeders
access to the full potential of marker-assisted
selection and introgression programs.
44
ANIMAL GENOME AND GENETIC MECHANISMS
Human hereditary disorders with identified
mutations and associated phenotype in nonrodent
species. (OBrien, S et al (1999) 286, 458
481) Animal Trait Human locus Cow
Uridine monophosphate synthetase deficiency
UMPS 3q13 Bta1 Leucocyte adhesion deficiency
(LAD) ITGB2 21q22.3 Bta1 Double muscling
GDF8 2q32.1 Bta2 Sheep Chondrodysplasia
(Spider Lamb) FGFR3 4p16.3 Oar Pig Porcine
stress syndrome on malignant hyperthemia RYR1
19q13.1 Ssc Hypercholesterolemia LDLR
19p13.2-13.1 Ssc Oedema disease FYT1
19q13.3 Ssc Coat color (dominant white) KIT
4p11-q12 Ssc Coat color (red/black) MC1R
16q24.3 Ssc
45
Milk Genomics
  • Use comparative information from HUGO to
    establish a functional genomics program to
    determine the molecular basis for the nutritional
    value of milk and milk components to juveniles
    (human and animal)
  • Assemble the set of tools necessary to poll the
    human genome and obtain the subset of the overall
    genome that is responsible for milk.
  • Apply to bovine genomics and existing knowledge
    of the bovine genome
  • Determine those genes that are induced by
    lactation within mammary epithelial cells
  • Relate 'milk genes' to their nutritional
    functions and nutritional value to humans as food
    components.
  • Bruce German/Juan Medrano/Wasyl Malyj

46
Genomics Center
  • Some of the 25 new genomics faculty will belong
    to the UC Davis Genome Center, the first new
    product of the initiative. (20m set aside for
    faculty)
  • Designed to establish the campus as an
    international leader in functional and
    comparative genomics, the center will include
    scientists specializing in gene studies from a
    multitude of disciplines, including human and
    animal medicine, engineering, agriculture,
    mathematics and the biological and physical
    sciences.
  • The Genome Center will also include a revitalized
    pharmacology and toxicology department in the
    School of Medicine and a group of bioinformatics
    faculty members who will provide the
    computational biology and informatics research
    needed to analyze the enormous amounts of data
    generated by the genomics research.

47
Examples of Biotech/IT Fusion Technologies
  • Genomics, proteomics and bioinformatics
  • Combinatorial chemistry
  • Peptide libraries- tea bags, beads
  • Combinatorial -biology
  • Directed evolution
  • DNA Shuffling, Molecular Breeding
  • High throughput analysis
  • Nucleic Acid based
  • Sequencing
  • Microarrays
  • Photolithography
  • Mirrors
  • Spotted Chips
  • Semi-conductor
  • Protein based
  • 2-D, electrospray/nanospray MS MALDI-TOF,
    LC/MS/MS, SELDI

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Expression Technologies
  • There are currently four commonly used approaches
    to high throughput, comprehensive analysis of
    relative transcript expression levels.
  • The enumeration of expressed sequence tags (ESTs)
    from representative cDNA libraries. A method of
    approximating the relative representation of the
    gene transcript within the starting cell
    population.
  • Serial Analysis of Gene Expression.The
    enumeration of serially concatenated 9-11 base
    tags from specially prepared cDNA libraries. The
    frequency of particular transcripts within the
    starting cell population is reflected by the
    number of times the associated sequence tag is
    encountered within the sequence pop
  • Differential Display Approaches Fragments
    defined by specific sequence delimiters can be
    used as unique identifiers of genes, when coupled
    with information about fragment length or
    fragment location within the expressed gene.
  • Array-based hybridization Based on the exquisite
    specificity of nucleotide interactions
    oligonucleotides or cDNA can be used to
    selectively identify or capture DNA or RNA of
    specific sequence composition.

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DNA Chips Competing and Complementary
Technologies
Technology
Advantages Over DNA Chips
Disadvantages Compared to DNA Chips
Description
Southern and northern blots
Standard techniques for identifying DNA sequences
and for studying changes in gene expression.
Technique can be optimized for each hybridization
event.
Slow, laborious, and low-throughput.
Microsphere array technology
Competing technology that immobilizes DNA to the
surface of coded beads instead of to the surface
of chips.
Hybridization occurs in solution, which increases
the rate in addition the quality control is
easier because aliquots of a larger batch can be
tested.
The density is not as high as most DNA chips in
addition the technology is not as widely accepted
as DNA chips.
Complementary technology in which mRNA pools
between two tissue samples, such as diseased
versus healthy, are matched up, and the
differentially expressed genes are subtracted out.
Subtraction libraries or differential display
technologies
These methods are laborious and insensitive.
Differences in gene expression are discovered
without knowing anything about the genes in
advance.
Serial Analysis of Gene Expression (SAGE)
Complementary technology in which sequence tags
are used to measure levels of expression.
More comprehensive technique for studying gene
expression changes.
This method is also laborious and insensitive.
The key advantages of DNA chips are their
throughput and ease of use.
21
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Base Pairs in GenBank
52
Whats Really Next
  • The post-genome era in biological research will
    take for granted ready access to huge amounts of
    genomic data.
  • The challenge will be understanding those data
    and using the understanding to solve real-world
    problems

53

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  • The two technologies that will shape the next
    century are biotechnology and information
    technology
  • Bill Gates
  • The two technologies that will have the
    greatest impact on each other in the new
    millennium are biotechnology and information
    technology
  • Martina McGloughlin
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