Transgenic animals

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Transgenic animals

  1. 1. Using DNA Technology to Produce Transgenic Animals Sherry Fuller-Espie, Ph.D., DIC Associate Professor, Cabrini College © Sherry Fuller-Espie, 2003
  2. 2. Transgenic Animals
  3. 3. Transgenic Animals: A Focus on Transgenic Mice Studies http:// www.hku.hk/biochem/tgcentre/transcentre.html
  4. 4. I. Introduction <ul><li>Transgenic animals: </li></ul><ul><ul><li>Animals which have been genetically engineered to contain one or more genes from an exogenous source. </li></ul></ul><ul><ul><li>Transgenes are integrated into the genome. </li></ul></ul><ul><ul><li>Transgenes can be transmitted through the germline to progeny. </li></ul></ul><ul><ul><li>First transgenic animal produced = “Founder Animal” </li></ul></ul>
  5. 5. II. Introduction of foreign genes into intact organisms <ul><li>Procedure is basically the same regardless of which animal is involved. </li></ul><ul><li>Integration usually occurs prior to DNA replication in the fertilized oocyte. </li></ul><ul><ul><li>Majority of transgenic animals carry the gene in all of their cells, including the germ cells. Transmission to next generation requires germline integration. </li></ul></ul><ul><ul><li>Some integration events occur subsequent to DNA replication giving rise to mosaic animals which may or may not contain the transgene in its germline. </li></ul></ul>
  6. 6. III. Procedure for Producing Transgenic Mice <ul><li>Three different breeding pairs of mice are required. </li></ul>
  7. 7. <ul><li>First Breeding Pair: </li></ul><ul><ul><li>Fertile male + superovulated female </li></ul></ul><ul><ul><ul><li>Fertile male = stud (changed regularly to ensure performance) </li></ul></ul></ul><ul><ul><ul><li>Superovulated female = immature female induced to superovulate </li></ul></ul></ul><ul><ul><ul><ul><li>Pregnant mare’s serum (=FSH) on day 1 </li></ul></ul></ul></ul><ul><ul><ul><ul><li>Human Chorionic Gonadotropin (=LH) on day 3 </li></ul></ul></ul></ul><ul><ul><ul><li>Mated on day 3 </li></ul></ul></ul><ul><ul><ul><li>Fertilized oocytes microinjected on day 4 with foreign DNA construct. </li></ul></ul></ul><ul><ul><ul><li>Microinjected oocytes are transferred to the oviducts of surrogate mothers at end of day 4. </li></ul></ul></ul>
  8. 8. <ul><li>Second breeding pair: </li></ul><ul><ul><li>Sterile male + surrogate mother </li></ul></ul><ul><ul><ul><li>Sterile male produced through vasectomy </li></ul></ul></ul><ul><ul><ul><li>Surrogate mother must mate to be suitable recipient of injected eggs </li></ul></ul></ul><ul><ul><ul><li>Mated on day 3 </li></ul></ul></ul><ul><ul><ul><li>Microinjected oocytes from first breeding pair are transferred to oviducts on day 4 </li></ul></ul></ul><ul><ul><ul><li>Embryos implant in uterine wall and are born 19 days later. </li></ul></ul></ul><ul><ul><ul><li>Southern blotting techniques confirm presence and copy number of transgenes. </li></ul></ul></ul>
  9. 9. <ul><li>Third breeding pair: </li></ul><ul><ul><li>Foster parents </li></ul></ul><ul><ul><ul><li>Fertile male + female mated to give birth on same day surrogate mother </li></ul></ul></ul><ul><ul><ul><li>Serves as foster parent if caesarian section is required for surrogate mother </li></ul></ul></ul>
  10. 10. <ul><li>http:// www.itba.mi.cnr.it/human_g ... transgenic.html </li></ul>
  11. 11. IV. Manipulation of Fertilized Oocytes See Slides
  12. 12. V. Gene Expression in Transgenic Mice <ul><li>In order to discriminate the products of the injected gene from those of an endogenous counterpart, the injected gene must be marked in some way. </li></ul><ul><ul><li>Mini-genes where exons are deleted of cDNA where introns are absent. </li></ul></ul><ul><ul><li>Modification by insertion/deletion/mutagenesis of a few nucleotides (e.g. the gain or loss of a restriction endonuclease site). </li></ul></ul><ul><ul><li>Hybrid genes where foreign epitopes are expressed on transgenic products. </li></ul></ul>
  13. 13. VI. Tissue-Specific Gene Expression <ul><li>Generally, if a tissue-specific gene is expressed at all, then it is expressed appropriately, despite the fact that it has integrated at a different chromosomal location. </li></ul>
  14. 14. VII. Examples of Studies Utilizing Transgenic Mice <ul><li>“ Pharm” animals (transgenic livestock) </li></ul><ul><ul><li>Bioreactors whose cells have been engineered to synthesize marketable proteins </li></ul></ul><ul><ul><li>DNA constructs contain desired gene and appropriate regulatory sequences (tissue-specific promoters) </li></ul></ul><ul><ul><li>More economical than producing desired proteins in cell culture </li></ul></ul>
  15. 15. <ul><li>Naked human Hb from pigs </li></ul><ul><li>Human lactoferrin in cows’ milk </li></ul><ul><li>Alpha-1-antitrypsin in sheep </li></ul><ul><li>HGH in mouse urine (uroplakin promoters) </li></ul><ul><li>Human antibodies in mice (H and L chain tgenics  hybridomas) </li></ul><ul><li>CfTCR in goats </li></ul><ul><li>Tissue plasminogen activator (TPA) in goats </li></ul><ul><li>Human antithrombin III in goats </li></ul><ul><li>Malaria antigens in goats (vaccine) </li></ul><ul><li>Alpha-glucosidase in rabbits (Pompe’s disease </li></ul>
  16. 16. VIII. Transgenic Pigs for the Production of Organs for Transplantation <ul><li>Pig organs are rejected acutely due to the presence of human antibodies to pig antigens. </li></ul><ul><li>Once human antibodies are bound to pig organs, human complement is activated and triggers the complement cascade and organ destruction. </li></ul><ul><li>Transgenic pigs with complement inhibitors have been produced and are gaining feasibility as a source of xenogeneic organs for transplantation. </li></ul>

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