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Technical blocks to efficient pig cloning

R.S. Prather

Division of Animal Science, National Swine Resource and Research Center, University of Missouri, Columbia, MO, United States

Genetic engineering of pigs relies heavily upon cloning by somatic cell nuclear transfer. The efficiency of pig cloning is 0.1 to 5% for those cell lines that work. Cloning by nuclear transfer has numerous technical inefficiencies as well as biological inefficiencies. The process involves eight main procedures: 1. Oocyte maturation, 2. Donor cell culture, 3. Oocyte enucleation, 4. Cell fusion or injection, 5. Oocyte activation, 6. Embryo culture, 7. Embryo transfer, and 8. Delivery of the offspring. The character of the inefficiencies that arise with each of these procedures will be discussed as well as possible solutions. Finally, some strategies to deal with the biological inefficiencies will also be addressed. While there is much room for improvement of the efficiencies, since there are numerous biological unknowns improvements in any one step may not result in dramatic changes in the birth of live offspring. Even with the low efficiencies we have produced over 530 live born cloned pigs and knocked out GGTA1, SIGLEC1, CFTR, SMN, and eGFP; knocked in deltaF508CFTR; and added transgenes for eGFP, DAF, CD55, CD59, ENTPD1, THBD, p23hRHO, hFAT-1, NOS3, CAT, INS, SMN2, FIX, FVIII, VWF, FURIN, SERPINA1, TK1, CDA, CFTR, and eGFP-PSMA1. Additional modifications are underway and, while the procedures are inefficient, new models are being successfully created. Funding from the National Institutes of Health and Food for the 21^st Century.