Animal cloning

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Animal cloning

  1. 1. ANIMAL CLONINGBLS 308/209HOZA, A.SMAY, 2009<br />
  2. 2. Types of cloning<br />• Recombinant DNA cloning<br />• Therapeutic cloning<br />– Stem cells<br />• Reproductive cloning<br />– Embryo splitting<br />– Somatic cell nuclear transfer (SCNT)<br />
  3. 3. History of cloning by SCNT<br />• Nuclear transfer was performed with frogs in the 1950s<br />• Cattle were cloned by transfer of embryonic nuclei in the 1980s<br />• Dolly was the first animal cloned by SCNT from an adult cell in 1997<br />
  4. 4. Animals cloned by SCNT<br />• Sheep, pig, goat, cattle, cat, rabbit, mouse, mule, horse, rat, deer, fish<br />• Cloning efficiency for Dolly, 1/277 reconstructed embryos (0.3%)<br />
  5. 5. Basic SCNT Methodology<br />Injection of donor cell under the ZonaPellucida<br />Injection of donor cell or Isolated nucleus into The cytoplasm<br />
  6. 6. SCNT-chromatin transfer<br />• Chromatin condensation<br />• Fusion with an enucleated egg<br />• Enhanced survival of clones<br />
  7. 7. Challenges to successful SCNT<br />• Reprogram a nucleus from a differentiated stage (somatic cell) to an embryonic stage<br />• Properly activate genes necessary for early embryonic development and suppress differentiation associated genes<br />
  8. 8. Pet cloning<br />• Dog cloning- MissiplicityProject<br />• Cat cloning<br />Shin et al., 2002<br />
  9. 9. Clones of clones<br />• Mice have been serially cloned to 6 generations<br />• Cattle have been serially cloned to 2 generations (3rd generation failed)<br />Donor<br />clone 1<br />clone 2<br />Kubota et al, 2004<br />
  10. 10. Literature survey of developmental problems in cloned animals<br />77% of cloned<br />Animals are<br />healthy<br />Cibelli, 2002<br />
  11. 11. Embryonic problems with SCNT<br />• High mortality during gestation<br />• >65% of one cell cloned embryos fail to develop to morula/blastocyst<br />• Days 30-60<br />– Cloned cattle: 50-100% embryonic loss<br />– Normal service: 2-10% loss<br />– In vitro fertilization: 16% loss<br />• Increased incidence of spontaneous abortions during second trimester<br />• Abnormal placentas<br />
  12. 12. Postnatal problems with SCNT<br />• Abnormally high birth weight<br />• Respiratory and metabolic abnormalities<br />– Lung dysmaturity<br />– Pulmonary hypertension<br />• “Adult clone sudden death syndrome” -<br />cloned pigs died of heart failure at less than6 months <br />
  13. 13. Factors contributing to early embryonic death<br />• Extensive micromanipulation of oocytes and<br />somatic cells<br />– Enucleation of an oocyte, removal of 5-15% of ooplasm<br />– Electrical induced fusion<br />• Incompatibility between mitochondrial and nuclear DNA<br />• Incomplete reprogramming of somatic nucleus<br />• Immunological rejection of SCNT fetuses -<br />abnormal MHC-1 expression by trophoblast cells<br />
  14. 14. Early death of cloned mice<br />Ogonuki et al., 2002<br />
  15. 15. Abnormal expression of genes in cloned mice<br />• Global analysis of gene expression in cloned mice using DNA microarrays<br />Humphreys et al., 2002<br />
  16. 16. Cloned mice<br />• Cloned mice have an obese phenotype<br />• Phenotype is not transmitted to offspring<br />• Obesity due to epigenetic changes<br />Tamashiro et al., 2002<br />
  17. 17. Premature ageing of clones?<br />• Telomeres are specialized structures<br />at the ends of linear chromosomes that shorten with age<br />• Cloned cattle have widely varying telomere lengths<br />• Dolly’s telomeres<br />– shorter than an age-matched control<br />– consistent with a 6-year old mammary<br />cell<br /> telomere<br />
  18. 18. Advantages to SCNT<br />• Restore endangered species<br />• Allows for the targeted deletion (knockout) of genes in farm animals<br />• Alternative method for generating transgenic animals as bioreactors (pharming)<br />
  19. 19. Cloning of endangered species<br />• Gaur bull calf cloned using a domestic cow embryo and surrogate mother<br />• Cells from skin cells frozen for 8 years<br />• Clone died 48 hours after birth<br />Lanza et al., 2000<br />
  20. 20. Cloning of endangered species<br />• Cloned African wildcat<br />• Nucleus from frozen cell transferred into domestic cat oocyte<br />
  21. 21. Xenotransplantation<br />• Transfer of cells, tissues or organs between species<br />• Pigs contain alpha galactosyl sugars on the cell surface.<br />• Human and higher primate lack alpha-galactosyl sugars; thus have antibodies to alpha-gal<br />• Alpha-gal antibodies lead to acute tissue rejection<br />
  22. 22. Gene knockout technology<br />• Combine SCNT with somatic cell gene knockout technology<br />Kent-First and First, 2000<br />
  23. 23. Gene knockout in farm animals<br />• Cloned pigs lacking the alpha-galactosyltransferase gene - xenotransplantation<br />• Cloned cattle lacking prion gene <br />
  24. 24. Transgenic animal bioreactors<br />• Produce important human pharmaceutical proteins in milk<br />• Clones would provide a genetically stable line for protein production<br />
  25. 25. Advantages to cloning transgenic animals as bioreactors<br />• Increase efficiency to 100% starting with a transgenic cell line<br />• Can predetermine sex<br />• Allows for rapid expansion of genetically identical animals<br />
  26. 26. Future cloning issues<br />• Food consumption risks of cloned animals<br />– Health of animal clones<br />– Composition of meat and milk<br />• No differences in milk composition were seen between clones and control dairy cattle<br />– total solids, fat, lactose, and protein (Norman and Walsh, 2004)<br />
  27. 27. • Many different species have been cloned<br />• Animal cloning has many successes but also many complications<br />• Refinement of technique will overcome some of these problems<br />• Cloning Applications<br />– transgenesis<br />– gene knockout<br />

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