7. Gene transfer methods Microinjection of recombinant DNA into the male pronucleus of an in vitro fertilized egg. 2. Embryonic stem cell transfer (ES). Other methods: Chemical or 2. Viral delivery into ES cells, or homologous recombination with ES cells.
8. Microinjection • InjectDNA molecules (transgenes) directlyinto male pronucleus • Most popular technology, commercial available • Success rates range from 10-30% depending on skills and constructs •Efficiency is not related to the copies of transgenesinjected
9. Microinjection • The technique can be applied to other species • No theoretical limit for the size of the construct • Overall efficiency is still low, particularly for farm animals • Tandem repeat of gene constructs (head-tail) • High frequency of mosaic • Initial investment is high
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12. Virus mediated gene transfer • Earliest method for successful gene transfer in mammals • Virus has transfection property • Killed virus is replication defective • The virus gene is replaced with transgene gene • The transgene is delivered to the host cell by transfection(gene therapy) • Can be used to transfect a wide range of cells, e.g., ES cells
13. Virus mediated gene transfer •Direct transfection of embryos has resulted nongermlinetransgenics • ES cells transfection has resulted in germlinetransgenics • Has succeeded in chickens and fish • Transfectingoocytes resulted in 100% transgenics • Only small transgene construct is usable (8 kb or less) • More research is needed on the safety of the method
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15. ES cells • May be transfected with transgene or with genes removed (knockout) or inserted prior to microinjection • Has revolutionized genetics, development, immunology and cancer research in mice
17. Approaches to using ES cells to create transgenic animals. The transgenecan be microinjected into the ES cells can be introduced by a virus, Chemical (e.g calcium phosphate or rubidium chloride by using homologous recombination.
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19. Nuclear transfer • Creation of Dolly • Somatic cells be transfected, or genetically altered prior to NT • 100% efficiency of any progeny • Low efficiency • Abnormal development
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21. Screening for Transgenic Positives • Identification of transgene integration - DNA • Detect transgene transcription - mRNA • Detect transgene expression - protein
25. The transgenic procedure is promising, but very expensive, and still has a low success rate especially for larger farm animals.
26. A mouse engineered in 1987 to express the clot dissolver drug tissue plasminogen activator (tPA)
27. In 1990, human alphaantitrypsin, an inhibitor used to treat emphysema, was produced in the mouse’s milk.
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29. E.g6 transgenic lambs for Roslin Institute - created in 1997 to produce a human clotting factor in their milk.
30. The first transpharmer goats were produced in 1991 at the Tufts University School of Veterinary Medicine to produce tissue plasminogen activator, a clotdissolving drug.
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32. Two calves were produced by microinjection of DNA into embryos that were then implanted in surrogate mothers and born alive.
33. One of these cows was female the transgene rearranged itself so that a portion of the lactoferrincDNA was deleted.
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38. open doors past what we previously have known to cure diseases!!??
39. possibly end world hunger entirely!!!??Transgenic Art - Creating monsters!!! E.g“Alba,” the rabbit that glows under UV light!!!!???
40. Eduardo “transgenic art.” refers to animals and plants with a planned genome intended to express an artistic idea symbolized by the proteins they code for.
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43. Applications of transgenic farm animals • Agricultural applications • Bioreactors • Organ/cell/tissue donors • Basic research/disease model
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