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Transgenic creatures and knockout creatures

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  1. Transgenic animals and knockout animals

  2. 3 main ways to do biological research: • Do research in test tubes. • Do research with cells. • Do research directly with animals.

  3. Transgenic animals and knockout animals Part 1: Transgenic animals: • Introduction to transgenic animals. • How to make transgenic animals? • How to make conditional transgenic animals? • Applications of transgenic animals. Part 2: Knockout animals • Introduction to knockout animals. • How to make knockout animals? • How to make conditional knockout animals? • Applications of knockout animals.

  4. Transgenic Animal • Animal hasone or more foreign genes inserted into chromosome DNA inside its cellsartificially. • After injecting foreign gene into the pronucleus of a fertilized egg or blastocyst, foreign gene is inserted in a random fashion into chromosome DNA: • Randomly (Foreign gene may disrupt an endogenous gene important for normal development, and the chance is about 10%. ) • multiple copies

  5. Transgenic animals and knockout animals Part 1: transgenic animals: • Introduction to transgenic animals. • How to make transgenic animals? • How to make conditional transgenic animal? • Applications of transgenic animals. Part 2: Knockout animals • Introduction to knockout animals. • How to make knockout animals? • How to make conditional knockout animals? • Applications of knockout animal.

  6. ES cell transformation Injection of gene into fertilized egg

  7. Method 1: ES cell transformation vs. Method 2: Injection of gene into fertilized egg 1. ES cell transformation works well in mice only. Other transgenic animals are produced by egg injection 2. ES cell transformation provides more control of the integration step (selection of stably transfected ES cells) 3. Injection of gene into fertilized egg is less reliable (viability of eggs, frequency of integration), but it helps to avoids chimeric animals

  8. Injecting fertilized eggs • The eggs are harvested from mice (superovulated or natural matings). • The DNA is usually injected into the male pronucleus. • The eggs can be transferred in the same day (1 cell) or the next day (2-cells) into pseudopregnant female oviducts.

  9. Breeding Transgenic animals(transgenic founders) • Transgenic animals Individually are backcrossed to non-transgenic animals. • DO NOT intercross different founders.Each founder results from a separate RANDOM transgene integration event.

  10. Transgenic animals and knockout animals Part 1: transgenic animals: • Introduction to transgenic animals. • How to make transgenic animals? • How to make conditional transgenic animals? • Applications of transgenic animals. Part 2: Knockout animals • Introduction to knockout animals. • How to make knockout animals? • How to make conditional knockout animals? • Applications of knockout animal.

  11. Conditional Transgenic mouse The expression of transgene in transgenic mouse can be induced

  12. Important Considerations for Conditional Transgenes • Transgenes have low or no expression when not induced • Large difference between induced and non-induced gene expression • Transgene expression rapidly turns on or off. • Inducer (doxycycline, tamoxifen, cre) is not toxic and easily administered

  13. Tetracycline Controlled Transactivator tTA “Tet-off” tetR VP16 Doxycycline blocks tTA DNA binding tTA binds to tetO to activate transcription

  14. Reverse Tetracycline Controlled Transactivator tTA “Tet-on” rtetR VP16 Doxycycline allows rtTA to bind to tetO Without doxcycline rtTA can not bind to tetO

  15. Tetracycline Regulation: Summary No Doxycycline Doxycycline tTAexpressednot expressed rtTAnot expressedexpressed

  16. Transgenic animals and knockout animals Part 1: transgenic animals: • Introduction to transgenic animals. • How to make transgenic animals? • How to make conditional transgenic animal? • Applications of transgenic animals. Part 2: Knockout animals • Introduction to knockout animals. • How to make knockout animals? • How to make conditional knockout animals? • Applications of knockout animal.

  17. Applications of Transgenic Animals Transgenic mice are often generated to 1. characterize the ability of a promoter to direct tissue-specific gene expression e.g. a promoter can be attached to a reporter gene such as LacZ or GFP 2. examine the effects of overexpressing and misexpressing endogenous or foreign genes at specific times and locations in the animals 3 Study gene function Many human diseases can be modeled by introducing the same mutation into the mouse. Intact animal provides a more complete and physiologically relevant picture of a transgene's function than in vitro testing. 4. Drug testing

  18. Example 1: Transgenic Cattle • Cloned transgenic cattle produce milk with higher levels of beta-caein and k-casein Published in Nature, Jan, 2003

  19. Example 2: Transgenic Mouse The growth hormone gene has been engineered to be expressed at high levels in animals. The result: BIG ANIMALS Mice fed with heavy metals are 2-3 times larger Metallothionein promoter regulated by heavy metals

  20. Example 3: Transgenic Mouse Trangenic mouse embryo in which the promoter for a gene expressed in neuronal progenitors (neurogenin 1)drives expression of a beta-galactosidase reporter gene. Neural structures expressing the reporter transgene are dark blue-green.

  21. Example 4: GFP transgenic mouse (Nagy) 9.5 day embryos - GFP and wt Tail tip

  22. GFP transgenic mouse (Nagy)

  23. Example 5: Wild and domestic trout respond differently to overproduction of growth hormone. So, GH is not effective to domestic trout.

  24. Example 6: Transgenic mice as tools • Normal mice can't be infected with polio virus. They lack the cell-surface Polio virus receptor. But, human has Polio virus receptor. • Transgenic mice expressing the human gene for the Polio receptor can be infected by polio virus and even develop paralysis and other pathological changes characteristic of the disease in humans

  25. Transgenic animals and knockout animals Part 1: transgenic animals: • Introduction to transgenic animals. • How to make transgenic animals? • How to make conditional transgenic animal? • Applications of transgenic animals. Part 2: Knockout animals • Introduction to knockout animals. • How to make knockout animals? • How to make conditional knockout animals? • Applications of knockout animals.

  26. knock-out Animal One endogenous gene in an animal is changed. The gene can not be expressed and loses its functions. • DNA is introduced first into embryonic stem (ES) cells. • ES cells that have undergone homologous recombination are identified. • ES cells are injected into a 4 day old mouse embryo: a blastocyst. • Knockout animal is derived from the blastocyst.

  27. Transgenic animals and knockout animals Part 1: transgenic animals: • Introduction to transgenic animals. • How to make transgenic animals? • How to make conditional transgenic animal? • Applications of transgenic animals. Part 2: Knockout animals • Introduction to knockout animals. • How to make knockout animals? • How to make conditional knockout animals? • Applications of knockout animals.

  28. Vector design • Recombinant DNA methods: Simple KO • Structural gene desired (e.g. insulin gene) to be "knocked out" is replaced partly or completely by a positive selection marker to knock out the gene functions. • Vector DNA to enable the molecules to be inserted into host DNA molecules

  29. KNOCKOUT MICE Isolate gene X and insert it into vector. Inactivate the gene by inserting a marker gene that make cell resistant to antibiotic (e.g. Neomycin) Normal (+) gene X Genome Defective (-) Gene X Transfer vector with (-) gene X into ES cells (embryonic stem cells) VECTOR e.g.(NeoR) MARKER GENE

  30. Vector and genome will recombine via homologous sequences Genomic gene Exon 4 Exon 2 Exon 3 Exon 1 Homologous recombination and gene disrution Grow ES cells in antibiotic containing media; Only cell with marker gene (without normal target gene) will survive

  31. Solution: Replacement vectors The knock-out construct contains the 1) NeoR gene flanked by 2) two segments of the target gene and 3) the HSVtk gene Part of the gene replaced with NeoR ES cells are selected for integration of NeoR and against integration of HSVtk* (NeoR+/ HSVtk-) on gancyclovir Problems with homologous recombination Unwanted random non-homologous recombination is very frequent. This method provides no selection against it

  32. Homologous recombination Random integration NeoR NeoR+/ HSVtk- NeoR+/ HSVtk+ HSVtk will convert gancyclovir into a toxic drug and kill HSVtk+ cells Replacement vectors Gene segment 1 Gene segment 2 NeoR Linearized replacement plasmid HSVtk

  33. Typical KO vector *tk:thymidine kinase

  34. Inject ES cells with (-) gene X into early mouse embryo Transfer embryos to surrogate mothers Resulting chimaras have some cells with (+) gene X and (-) gene X. Mate them with normal mice It is lucky, if germline contain (-) gene X Screen pups to find -/+ and mate them Next generation will split as 3:1 (Mendelian)

  35. Embryonic stem cells • Harvested from the inner cell mass of mouse blastocysts • Grown in culture and retain their full potential to produce all the cells of the mature animal, including its gametes.

  36. ES cells growing in culture

  37. ES cells are transformed • Cultured ES cells are exposed to the vector • Electroporation punched holes in the walls of the ES cells • Vector in solution flows into the ES cells • The cells that don't die are selected for transformation using the positive selection marker • Randomly inserted vectors will be killed by gancyclovir

  38. Successfully transformed ES cells are injected into blastocysts

  39. Implantation of blastocysts • The blastocysts injected with transformed ES cells are left to rest for a couple of hours • Expanded blastocysts are transferred to the uterine horn of a pseudopregnant female • Max. 1/3 of transferred blastocysts will develop into healthy pups

  40. Implanting blastocysts 1 2

  41. Implanting blastocysts 3 4

  42. Testing the offspring • A small piece of tissue - tail or ear - is examined for the desired gene • 10-20% will have it and they will be heterozygous for the gene

  43. Breeding Chimeras (knock-out founder) Chimera - the founder • germ-line transmission - usually the ES cells are derived from a 129 mouse strain (agouti or white colour) and the ES cells are injected into blastocyst derived from a C57Bl/6 mouse (black). • The more that the ES cells contribute to the genome of the knockout mouse, the more the coat colour will be agouti. The chimera mouse is usually “tiger” striped.

  44. Breeding Chimeras (knock-out founder) • Males that are 40% to 100% based on agouti coat colour should be bred • Females should not be bred (low incidence of success). • Breed aggressively- rotate females through male's cage. If the male produces more than 6 litters without transmitting knockout gene, the knockout gene will not likely go to germline and should not be used for more breeding.

  45. Littermates Black mouse - no apparent ES cell contribution Chimeric founder - strong ES cell contribution Chimeric founder - weaker ES cell contribution