1. ANIMAL CLONINGBLS 308/209HOZA, A.SMAY, 2009

2. Types of cloning • Recombinant DNA cloning • Therapeutic cloning – Stem cells • Reproductive cloning – Embryo splitting – Somatic cell nuclear transfer (SCNT)

3. History of cloning by SCNT • Nuclear transfer was performed with frogs in the 1950s • Cattle were cloned by transfer of embryonic nuclei in the 1980s • Dolly was the first animal cloned by SCNT from an adult cell in 1997

4. Animals cloned by SCNT • Sheep, pig, goat, cattle, cat, rabbit, mouse, mule, horse, rat, deer, fish • Cloning efficiency for Dolly, 1/277 reconstructed embryos (0.3%)

5. Basic SCNT Methodology Injection of donor cell under the ZonaPellucida Injection of donor cell or Isolated nucleus into The cytoplasm

6. SCNT-chromatin transfer • Chromatin condensation • Fusion with an enucleated egg • Enhanced survival of clones

7. Challenges to successful SCNT • Reprogram a nucleus from a differentiated stage (somatic cell) to an embryonic stage • Properly activate genes necessary for early embryonic development and suppress differentiation associated genes

8. Pet cloning • Dog cloning- MissiplicityProject • Cat cloning Shin et al., 2002

9. Clones of clones • Mice have been serially cloned to 6 generations • Cattle have been serially cloned to 2 generations (3rd generation failed) Donor clone 1 clone 2 Kubota et al, 2004

10. Literature survey of developmental problems in cloned animals 77% of cloned Animals are healthy Cibelli, 2002

11. Embryonic problems with SCNT • High mortality during gestation • >65% of one cell cloned embryos fail to develop to morula/blastocyst • Days 30-60 – Cloned cattle: 50-100% embryonic loss – Normal service: 2-10% loss – In vitro fertilization: 16% loss • Increased incidence of spontaneous abortions during second trimester • Abnormal placentas

12. Postnatal problems with SCNT • Abnormally high birth weight • Respiratory and metabolic abnormalities – Lung dysmaturity – Pulmonary hypertension • “Adult clone sudden death syndrome” - cloned pigs died of heart failure at less than6 months

13. Factors contributing to early embryonic death • Extensive micromanipulation of oocytes and somatic cells – Enucleation of an oocyte, removal of 5-15% of ooplasm – Electrical induced fusion • Incompatibility between mitochondrial and nuclear DNA • Incomplete reprogramming of somatic nucleus • Immunological rejection of SCNT fetuses - abnormal MHC-1 expression by trophoblast cells

14. Early death of cloned mice Ogonuki et al., 2002

15. Abnormal expression of genes in cloned mice • Global analysis of gene expression in cloned mice using DNA microarrays Humphreys et al., 2002

16. Cloned mice • Cloned mice have an obese phenotype • Phenotype is not transmitted to offspring • Obesity due to epigenetic changes Tamashiro et al., 2002

17. Premature ageing of clones? • Telomeres are specialized structures at the ends of linear chromosomes that shorten with age • Cloned cattle have widely varying telomere lengths • Dolly’s telomeres – shorter than an age-matched control – consistent with a 6-year old mammary cell telomere

18. Advantages to SCNT • Restore endangered species • Allows for the targeted deletion (knockout) of genes in farm animals • Alternative method for generating transgenic animals as bioreactors (pharming)

19. Cloning of endangered species • Gaur bull calf cloned using a domestic cow embryo and surrogate mother • Cells from skin cells frozen for 8 years • Clone died 48 hours after birth Lanza et al., 2000

20. Cloning of endangered species • Cloned African wildcat • Nucleus from frozen cell transferred into domestic cat oocyte

21. Xenotransplantation • Transfer of cells, tissues or organs between species • Pigs contain alpha galactosyl sugars on the cell surface. • Human and higher primate lack alpha-galactosyl sugars; thus have antibodies to alpha-gal • Alpha-gal antibodies lead to acute tissue rejection

22. Gene knockout technology • Combine SCNT with somatic cell gene knockout technology Kent-First and First, 2000

23. Gene knockout in farm animals • Cloned pigs lacking the alpha-galactosyltransferase gene - xenotransplantation • Cloned cattle lacking prion gene

24. Transgenic animal bioreactors • Produce important human pharmaceutical proteins in milk • Clones would provide a genetically stable line for protein production

25. Advantages to cloning transgenic animals as bioreactors • Increase efficiency to 100% starting with a transgenic cell line • Can predetermine sex • Allows for rapid expansion of genetically identical animals

26. Future cloning issues • Food consumption risks of cloned animals – Health of animal clones – Composition of meat and milk • No differences in milk composition were seen between clones and control dairy cattle – total solids, fat, lactose, and protein (Norman and Walsh, 2004)

27. • Many different species have been cloned • Animal cloning has many successes but also many complications • Refinement of technique will overcome some of these problems • Cloning Applications – transgenesis – gene knockout