introduction to animal biotechnology-2009new · the aims of producing transgenic animals...
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Introduction to AnimalBiotechnology
Introduction to Animal Science
Department of Animal ScienceNational Chung Hsing University
Pin-Chi Tang唐品琦
Office: 4C ; Lab: 204
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Reproductive Biotechnology
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Factors affecting reproductive efficiency
* Early development of the conceptus
* Genetic and environmental factors
* Nutrition
* Breeds
* Fertility and temperature
* Culling strategies
* Stress
* Effect of antibiotics and hormones
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Reproductive-biological procedureAI (artificial insemination)
Estrous synchronization
Regulation of parturition
ET (embryo transfer)Cryoperservation of gametes and embryos
Sexing of sperm and embryos
IVP (in vitro production of embryos)
Embryo bisection
NT (nuclear transfer)
Microinjection technology (Sperm, DNA, RNA...)
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5Applied Animal Reproduction, 6th ed., 2004
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6Pathways to Pregnancy and Parturition, 1st ed.,1997
子宮阜
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7Applied Animal Reproduction, 6th ed., 2004
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Estrus Detection
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9Pathways to Pregnancy and Parturition, 1st ed.,1997
Stages of the estrous cycle
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Cow Swine
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Artificial Insemination, AI
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Advantages of AI•Genetic improvement e.g., widespread use ofoutstanding sires; improving accuracy of selection throughprogeny test; permitting crossbreeding; introduction of newgenetics
•Availability of accurate breeding records
•Control of veneral diseases
•Economic service
•Safety through elimination of dangerous male
•Use of deep-frozen semen after a donor is dead
•Gender control
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Bull semen collection by artificial vagina
Bull semen collection by electrostimuation
Semen Collection & Artificial Insemination
Boar semen collection by massage
Swine insemination
Cow insemination
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Cryoperservation ofgametes and embryos
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Freezing injury
destroy cell function irreversibly
intracellular ice crystal formation andsalt deposits
changes in the composition of surroundingmilieu at thawing phase
cytotoxicity of cryoprotectants
Cryopreservation
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Cryoprotective agents* high water solubility; high permeability;
low toxicity* alcohols, amines, sugars and proteins* dimethyl sulfoxide (DMSO); 1,2-propanediol
(PROH); ethylene glycol (EG); glycerol
Antifreeze proteins* many animals and plants in nature use
cryoprotective agents* production of antifreeze proteins, mainly
glyopeptides
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Boar semen cooling
Bull semen cooling and freezing
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Embryo Transfer
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Cell transplantationMale gametes have potential ofdeveloping in a surrogate environment
Molecular breeders
Nuclear transferMammalian embryogenesisby asexual reproduction
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Transgenic Animals
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normal mouse transgenic mouse
Promoter: metallothionine-1
Transgene: rat GH
Science 1982, 385:810-813
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The Aims of producing transgenic animals
Biological, biomedical, veterinary and
genetic research
Agriculture: enhance growth and development
Increase disease resistance
Xenotransplantation
Produce foreign proteins
Gene therapy
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The Methods to produce transgenic animals
Microinjection
Retroviral vector infection
Sperm vector
Embryonic stem cell (ES cells):partical gun / transfection / electroporation
Nuclear transfer
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DNA microinjection
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Nuclear Transfer
Enucleation Nuclear transfer
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Improvement of agriculture traits
Dietary modifications of animal products
Environmentally friendly farm animals
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27Nature Biotech. 2001, 19: 741–745
Total phosphorus content (on a drymatter basis) of fecal matter fromnon-transgenic pigs ( ) andtransgenic pigs ( ) of line WAfed different levelsof soybeanmeal as the sole source ofdietary phosphorus.
DM, Dry matter content of feces.
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Transgenic animals in biomedicine
As models for human diseases
Physiol. Behav. 2001, 73:873-886.
AlzheimerAlzheimer’’s diseases disease
Amyloid precursor protein (APP)Presenilins (PS1 or PS2)
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HuntingtonHuntington’’s disease (HD)s disease (HD)
HD is caused by the expansion of cytosineadenine-guanine (CAG, translated into glutamine) trinucleotiderepeats in the first exon of the human huntingtin (HTT)gene.
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30Nature 2008, 453:921-924
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For pharmaceutical production
TRENDS in Biotech. 2004, 22:286-294.
> 2007Blood substituteGoatPhase I / IIVariousAbs
2008Hemophilia ACattlePhase IHAS
> 2008Cystic fibrosissheepExperimentalhFVIII
2007Lung emphysemaGoat / sheepPhase II / IIIa-AT
2006Dissolvingcoronary clots
GoatPhase II / IIITPA
2005Genetic heparinresistance
GoatPhase IIIAT III
Potentialmarketdate
Therapeuticapplication
Productionspecies
Developmentalphase
Protein
Proteins produced in the mammary gland of transgenic farm animals
AT III: antithrombin; TPA: tissue plasminogen; a-AT: antitrypsin; hFVIII; human clotting factor;Human serum albumin;
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32Adv. Biochem. Engin/Biotechnol. 2004, 91: 171–189
Procedure for generation of transgenic birds
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Genomics
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Genomics: the quantitative study of genes,
regulatory and noncoding sequences.
Transcriptomics: RNA and gene expression.
Proteomics: protein expression.
Metabolomics: metabolites and metabolic
networks.
etc......
Age of -omics
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Blueprint of Life
(Brooks et al., 2004)
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J. Watson and F. Crick
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Overview of gene expression
UAA Stop codon
Functional proteins
Post-translationalmodifications
(Brooks et al., 2004)
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Defining GenesA complete chromosomal segment
responsible for making a functional product
Open Reading Frame (ORF): a string of codonsbounded by start and stop signals.
Evidence of transcription: RNA or proteinexpression is a hallmark of a gene product.Methods: microarray, cDNA mapping, sequencingof EST clones, etc.
Sequence conservation: conserved DNAsequences suggest importance of the geneproduct.
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Genome
The entire collection of genesencoded by a particular organism.
Determination of an entire genomesequence is a prerequisite to understandingthe complete biology of an organism.
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Mitochondrial genome
•Animal has 15-17kb, no intron–Nearly always circular with 37 genes: 13
for proteins, 22 for tRNA and 2 for rRNA.
•Plant has 200kb-2Mb, with intron–51 genes, not too many more than animal.
–Besides introns, duplication of genes.
–Gain genes from chloroplast and losegene to nucleus.
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Genomics is the scientific study of structure,function and interrelationships of bothindividual genes and the genome in itsentirety (Bazer and Spencer, 2005)
Genomics
The field has evolved from identifying shortnucleotide strings of DNA to the sequencingof an organism’s complete genome.
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Structural genomics: construction ofsequence data and gene map.
Functional genomics: functions ofgenes, and their regulation and products.
Comparative genomics: comparegenes from different genomes toelucidate functional and evolutionalrelationship.
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功能性基因體的研究方法
• 基因表現全貌: EST 基因庫、差異展現法、雜合扣除法、 DNA microarray和proteome等
• 突變篩選: generation of random mutationin the genome, ENU, RNAi, T-DNA, EP-transposon, and so on…
• 轉基因或基因剔除: generation of targetmutation, knockout
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Current status of farm animal genomics
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45http://www.animalgenome.org/
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The meat industry can now use cow andchicken genomic data to confirm the quality ofmeat products. For example, meat producerscan now confirm the parentage of an Anguscattle breed by performing a genetic blood testor attempt to identify the SNPs associated withhigh-quality beef.
Using genomic information to determinedisease-resistant genes in shrimp and then areselectively mating the shrimp that carry them inorder to create disease resistant strains.
(Dove, 2005).
The potential of farm animal genomics
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In the healthcare arena, farm animal genomicwork will aid in enterprises such asxenotransplantation (the transfer of animal tissuesor organs into humans).
Though animal organs may be used someday tosatiate organ donor shortage, genomic work in thisarea is still in its early stage (Cascalho and Platt, 2001).
Many of the immediate practical applications offarm animal genomics show potential for growth inthis field.
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The database system is hosted at Roslin Institute aims toprovide a comprehensive public repository for genomemapping data from farmed and other animal species.
ArkDB is one of the major sources of information on farm animalgenomics.
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Applications of genomics in Animal Production
For decades, breeders have altered the genomes offarm animals by first searching for desired phenotypictraits and then selecting for superior animals tocontinue their lineage into the next generation (Harlizius
et al., 2004).
This genomic work has already facilitated areduction in genetic disorders in farm animals, asmany disease carriers are removed from breedingpopulations by purifying selection (Andersson, 2001).
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By studying diverse phenotypes over time,
researchers can now monitor mutations that
occur as wild species become domesticated(Andersson and Georges, 2004).
Farm animal food safety will remain a concern for
some time; however, advancements such as the
discovery of Escherichia coli resistant genes in the
pig (Edfors-Lilja, 1995) can mediate most of the problems.
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Moreover, resources devoted to investigating
the genomes of farm animals can bring
eventual economic benefits.
For example, isolation of DNA from animal tissue
can be used as an inexpensive method for
tracking the origin of a meat sample, providing
the recipient with the quality assurance of that
food (Brem, 2004).
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協助育種者選拔特定的主要突變(如大腸桿菌粘附抵抗力)或對抗負面的突變(像負面的鹵乙烷對偶基因或RN-對偶基因)。用於數量性狀的選拔(包含那些可以用傳統方法選拔的性狀),例如利用ESR之B對偶基因增加窩仔數及MC4R來降低飼料採食。增加選拔的準確性、容許對受性別限制性狀或銘印基因做選拔及容許選拔像肉質這類的性狀。
DNA分子資訊的應用
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基因晶片的發展與應用
數萬個基因或ESTs序列固定在一小塊面積的基材(即微陣列或基因晶片)上,例如載玻片,對某種生物材料進行研究,快速比對出mRNA具有差異表現的基因(Craig et al., 1990)。
利用DNA晶片或微陣列來檢測基因表現將提供動物性狀改良的新契機。
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Proteomics
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Protein Production Pathway(Central dogma of biology)
mRNA level expressed protein level nor does itindicate the nature of the functional protein product
GenomicSequence mRNA
ProteinProduct
FunctionalProteinProduct
TranscriptionalRegulation
TranslationalRegulation
Post-TranslationalRegulation
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(Chen et al., 2002)
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Definition of Proteome
The PROTEin complement expressed
by the genOME of an organism
(Wilkins, 1994; Wasinger et al., 1995)
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Definition of ProteomicsThe study of protein properties on a
global, integrated view of biologicalprocesses.
Expression Proteomics:Study of global changes in protein expression
Cell-map Proteomics:Study of protein-protein interactions through theisolation of protein complex
(Blackstock and Weir, 1999)
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Fields for proteomics related market
* Drug discovery* Diagnostics* Equipment supply* Protein/antibody chip
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Essential technology needed forproteomics
1. Separate large numbers of proteins
2. Identification of proteins
3. Study protein modifications
4. Progress from protein sequence to
3D structure and functions
5. Handle massive datasets- bioinformatics
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Key Technologies Driving Proteomics
Reproducible 2D Gel Technology
Staining and Scanning Technology
Mass Spectrometry for Identification
Databases (protein and genome)
Database Searching Algorithms
The key challenge in proteomic research is theautomation and integration of these technologies
(Winter, 2000)© 2008 Copyright SYHuang
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(Patterson and Aebersold, 2003)
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(Patterson and Aebersold, 2003)
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•normal samples
•diseased samples
•serum, CSF…
•± drug
•time-course
hexokinase
enolase
ORF YOR213p
Samples Separation Identification
1000s of samples=3 x1000s of gels
1000s of proteins/gel
IEF
MW
Digestion
106-1012 proteins
(Winter, 2000)
Experimental approach for proteomic studies
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70Key equipments necessary for proteomic studies.
MALDI TOFMALDI TOF/TOF
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(Fournier et al., 2007)
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Proteome databases on internet
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1. ExPASy (Expert Protein Analysis System)proteomics server
a. SWISS-PROT and TrEMBL- Protein sequencesb. PROSITE - Protein families and domainsc. SWISS-2DPAGE- Two-dimensional polyacrylamide gel
electrophoresisd. SWISS-3DIMAGE- 3D images of proteins and other
biological macromoleculese. SWISS-MODEL Repository- Automatically generated
protein modelsf. CD40Lbase- CD40 ligand defectsg. ENZYME- Enzyme nomenclatureh. SeqAnalRef- Sequence analysis bibliographic references
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2. Protein Data Bank: the ResearchCollaboratory for Structural Bioinformatics
3. The Protein Information Resource:International Protein Sequence Database
4. WORLD-2DPAGE: Index to 2-D PAGEdatabases and services
5. And many others
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Applications of proteomics
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Biomedicine (Clinical and Biomedical)
Drug discovery
Protein-protein interaction
Biological Application
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Clinical and BiomedicalApplication
*Clinical diagnosticsanalysis of body fluid and tissuemonitor disease processdiscover new disease markers
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* identify disease specific protein
* target validation and signal transduction
* drug mode-of-action
* formal drug toxicology
Drug discovery
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Biological Application of Proteomics(I)
* Tracking complexity# host-pathogen interaction
* Immunogenetic proteins# identify immuno-reactivity proteins# identify allergens
* Improve agricultural products# identify proteins important for disease protection# identify proteins related to economically
important traits© 2008 Copyright SYHuang
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* Value added agriculture products# dairy industry: seeking useful proteins# abattoir wastage: discovery of useful proteins# food industry: cereal protein replace casein
* Quality control# bring precision and definition to a new level of
protein-based products# identify genetic features of a cell line# detect the presence of a disease in biotechnology
products
Biological Application of Proteomics(II)
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