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Transgenesisprocess of introducing foreign or exogenousDNA into an animals genome
TransgeneDNA introduced
MiceCows
FishBirdsSheepGoats
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Improve genetic Features of domesticated Animals
Provide animal models for study of human diseases
Pharming
using farm animals for production of human pharmaceuticals-mammary glands
Study the genes regulation, development of animals
How to Get the Transgene Inserted Retroviral Vectors
MicroinjectionEmbryonic stem cells
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FIGURE 22.13
A. Remove eggsB. Fertilize in vitro C. DNA is microinjected into male pronucleus (prior to nuclear fusion)
100-1000 copies of gene
D. Implant eggs into surrogate
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2003 John Wiley and Sons Publishers
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FIGURE 22.8 (19.1)
Infect early stage embryo with replication-defective retrovirus
Limitationsonly small DNA insertsno regulatory sequencessafety
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FIGURE 19.4
Remove pluripotent ES cells from blastocyst Transfect ES [Site specific Integration - advantage] Selection Microinject back into blastocyst Implant
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1. Determine whether the progeny are TRANSGENICpiece of tail, PCR, Southern Blot
2. Mate this transgenic animal to determine whether thetransgene is in GERM line
3. Breed to get homogenous transgenic line
Integration of DNAG0 generation -Mosiacs(G 1 progeny)
all cells contain transgene
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Quiescent [G0] cells Plus EGG
First 3 cell division of Fertilized Eggno gene expressionembryo-initiating protein factors in
cytoplasm bind to embryonicdevelopment genes
When G0 nucleus is transferredcell specific regulatory proteins are strippedEIPF bind to EDG
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YACs contained many of these heavy and light chain segments
Knock out Mouse Segments, replace wuth Human segment genes
Fully human antibody made
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Applications
Increasing casein content of milk increase cheese productionLactose free milk (transgene lactase)Resistance to bacterial infectionsIn vivo immunization
transgene is specific Heavy and Light chain genes whichcreate mAn against a specific antigen
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Easy to purify - few other proteins in milk Doesnt harm transgenic animal - no change to physiology
rProtein is authentically modified post-translationally
Large quantities
Renewable source
Mammary Gland-specific Promoters
promoter and transcription termination sequences are present
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PIGSPST porcine somatotropin (growth hormone)
adverse effects- kidney, stomach, heart, sterility
human Hemoglobinto replace whole blood transfusions
SHEEPIncrease wool production
keratin promotergrowth factor
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Pr
Problem: Rejection
Antibodies from Host bind to Donor Organ
Elicits Inflammatory ResponseTransplanted Organ Lost
Solution:Transgene in Donor for Complement-Inhibiting Protein
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Birdstraditional methods can not be used
because of avian embryogenesis differencesno ES cells found
ALV resistant chickenstransgene - defective ALV genome
makes viral RNA and protein
but blocks assembly of wt viral particlesFish
aquaculturetransgene - growth hormone