Transgenic animals


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Transgenic animals

  1. 1. Transgenic animals and animal cloning Mohd bin Mahmud @ Mansor, FIAT
  2. 2. Introduction 95% of all transgenic animals are mice. 5% are rats, rabbits, goat, pigs, sheep, cows and fish. Among other species, why mice?
  3. 3. Introduction From mice, researchers now trying similar procedures on livestocks - as bioreactor Four routes to create transgenic mammals will be discussed The creation of therapeutic proteins
  4. 4. Transgenic Animals
  5. 5. Creation of transgenic animals 1. Microinjection of DNA 2. Integration of (retro) viral vector into an oocyte 3. Incorporation of genetically pluripotent stem cells into early embryo 4. Transfer of genetically altered nuclei into enucleated oocytes
  6. 6. Microinjection of DNA First reported in 1985 Most transgenic animals were created this way
  7. 7. Retrovirus vectors Replication defective retroviral vectors Replication competent retroviral vectors Incubating bovine oocytes in the final stage of maturation increase rate of transgenesis Cloning capacity is limited (<10kb
  8. 8. Pluripotent stem cells Capability to developed into many type of cells Can be maintained in tissue culture Genetically manipulated and selected Highly successful in mice, but not so much in other organisms
  9. 9. Nuclear transfer technology Somatic cloning Transfer of donor nucleus into cytoplasm of an enucleated zygote or oocyte Achieved in cattle, goats, pigs, rabbits, mulls, horses, cats, dogs and some wild life species
  10. 10. "Pharming" and human proteins 1970s, DNA manipulation provided a significant alternative source for many drugs made of protein • Human growth hormone: human cadaver • Insulin: slaughtered pigs Using DNA technology, insulin can be harvested from recombinant bacteria Overexpression of human genes in bacteria not always yield a functionally active protein The protein need to be post-translational modified - phosphorylated or glycosylated to become active
  11. 11. Transgenic in sheep, goats, cows and pigs The idea of expressing recombinant proteins into milk started in 1989 Since then until 2002, 25 types of human therapeutic have been produced in transgenic animals Biopharm animals Why we use animals as bioreactors rather than bacteria?
  12. 12. Transgenic in sheep, goats, cows and pigs Example: • Blood clotting factors (VIII and IX) • Anti thrombin III - intravascular coagulation • Collagen - burns and bone fractures • Fibrinogen - burns and after surgery • Human fertility hormones • Human serum albumin • Human hemoglobin • Lactoferrin (found in mother milk) • Tissue plasminogen activator
  13. 13. Transgenic in sheep, goats, cows and pigs Mostly are using sheep, goats, cows or pigs Regulated by FDA Enormous cost involved in production of a drug in livestock • US$ 800 million • Around 15 years • 7 years to generate transgenic livestock only
  14. 14. Transgenic in sheep, goats, cows and pigs Amount of blood clotting factor IX needed every year is 2kg/year Assuming animals produce 1g of protein per liter and purification efficiency is 30%, then a pig will produce 100g, sheep 125g, and a cow 3kg. So we only need one cow per country to have enough supply of blood clotting factor IX
  15. 15. Transgenic in sheep, goats, cows and pigs Cost estimating to produce one transgenic cow is around US$ 300,000 to US$500,00 There are also costs of failed experiments
  16. 16. Transgenic Chicken Laying eggs containing human protein in egg whites Basic strategy: target expression of a therapeutic protein to a protein secretory tissue (oviduct of a laying hens) using a regulatory sequences of one of the native proteins (ovalbumin) synthesized in that tissues.
  17. 17. Transgenic sheep for wool Other than production of human proteins Transgenic sheep with better wool Diet of high sulphur-containing amino acids Sheep in cysteine - sulphur rich that is an essential building block for keratin Mammal cannot produce their own cysteine - from gut dwelling bacteria that help them in digestion
  18. 18. Transgenic fish Atlantic salmon expressing growth hormone Approved for consumption 4-6 times the growth rate 10 - 20% improvement in feed conversion efficiency Shorter production time, reduced costs, improve profitability
  19. 19. Xenotransplantation "Procedure that involves the transplantation, implantation, or infusion into a human recipient of either (a) live cells, tissues or organs from nonhuman animal source or (b) human body fluids, cells, tissues, or organs that have had ex vivo contact with live nonhuman animal cells, tissues or organs"
  20. 20. Xenotransplantation Transgenic pigs • Provide organs (heart, kidneys) suitable for human transplantation • Gene that encodes a human cell-surface protein • Prevent components of human immune system from attacking and destroying the organs
  21. 21. Animal cloning The first cloned mammal - Dolly Cloned from a single adult, somatic cells taken from her mother's udder Unfertilized egg cell with the nucleus removed Fused with the cell from udder The fused cell then made to divide and develop into a normal embryo Since then, goats, mice, pigs,cats, rabbits, mules and horses. Will human be next?
  22. 22. Animal cloning In principle it is possible But actually the cloning efficiency is extremely low with only less than 4% embryos developing to live offspring. Offspring obtained from cloning also reported to experience early death or severe abnormalities Dolly was suffering from arthritis and died because of lung disease
  23. 23. Other stories Polly - human blood clotting factor IX Transgenic pigs - rich with omega 3
  24. 24. Creating transgenic animals
  25. 25. Designing the gene construct Sourcing the transgene Make a transgenic embryo Select +ve transgene cells Making the gene construct Transfecting bovine cells PCR Confirming the cow is transgenic Analyzing of protein expressed qPCR Fluorescence in situ hybridization
  26. 26. 1. Designing a construct Overall function Align it all logically Promoter, gene of interest, poly A tail Cell or tissue specific promoter
  27. 27. 1. Designing a construct Promoter Regulating the spatial and temporal expression pattern of a transgene Sequences are isolated from upstream regions of endogenous mammalian genes Possibility of being tissue specific and developmental stage specific Inducible promoter • Giving inducible expression
  28. 28. 1. Designing a construct Intron Leads to significantly greater transgene expression Effects on mRNA stabilization Efficient translocation from nucleus to cytoplasm Examples: rabbit beta-globin intron or simian virus 40 (SV40)
  29. 29. 1. Designing a construct Protein coding sequence Full length cDNA derived from RNA of a gene of interest Contains a translational start codon (ATG) and translational stop codon • So ribosome can properly scan and recognize the proper translation start and stop sites of mRNA Kozak sequence upstream of a start codon
  30. 30. 1. Designing a construct Poly A Promotes translation by ribosomes Protects the mRNA from nucleases
  31. 31. 1. Designing a construct Enhancer Transcription factor recruitment Release of RNA polymerase II
  32. 32. 2. Sourcing the transgene Provide by external institution Come up within a vector Together with restriction site Or synthetically constructed Given that you have the....
  33. 33. 3. Making the gene construct Just sticking the gene into an expression vector Vector backbone, opened by RE And gene of interest, cut with same RE Mix together all mixtures Incubation period may vary
  34. 34. 4. Transfecting bovine cells Introduction of DNA into a cell Cells growing in the dish Using some methods - chemically or physiologically Some example?
  35. 35. 5. Select positive bovine cells How do we do screening?
  36. 36. 6. Make a transgenic embryo Isolate egg from donor Remove the DNA Fuse the transgenic cells Incubated in the lab ~ 7 days Transfer into surrogate mother
  37. 37. 7 Confirming the cow is . transgenic Southern blotting • Low false positive rate • Many information can be obtained from the blot • PCR may be used for large sample (screen positive) but must be followed by SB atleast once • PCR is best for offspring qPCR Fluorescence in situ hybridization Protein analysis
  38. 38. Questions? Thank you.
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