The production of genetically identical animals by nuclear transfer carries importance to biomedical research as it eliminates genotypic variation allowing greater statistical validity with fewer experimental animals. Furthermore, fundamental investigations into the nature of the immune system would become feasible. We established a successful rhesus monkey in vitro fertilization (IVF) program at the Oregon Regional Primate Research Center where monkeys have been produced by nuclear transfer technology employing enucleated oocytes as recipient cytoplasts and individual blastomeres from IVF-produced embryos as nuclear donors. Current experiments are designed to improve the nuclear transfer procedure in rhesus macaques by identifying a source of donor nuclei which can be used to support the creation of large clone sizes. These are two specific aims (1) To optimize the donor nuclear source for the production of clonally-derived rhesus monkeys by nuclear transfer. Three possible sources of donor nuclei will be evaluated for the routine production of genetically identical animals, namely, individual blastomeres from IVF-produced embryos (our proven method), primary cultures of fibroblasts and immortalized embryonic stem (ES) cells. Initially, nuclei from fetal and adult fibroblasts and ES cells, transfected with green fluorescent protein to allow confirmation of donor nucleus participation in development, will be examined for their ability to support preimplantation development following nuclear transfer into Metaphase II cytoplasts. Experimentation will determine whether cell cycle staging is critical for successful reprogramming of the nucleus following transfer. (2) To produce clonally-derived rhesus monkeys. The optimal source of donor nuclei established under Aim 1 will then be employed in efforts to produce clonally-derived, transgeneic monkeys. Clones of 50 reconstituted embryos will be created with fibroblasts and with ES cells and stored frozen. Transfers involving 2 embryos of the same clone will be conducted in synchronized host mothers. IVF-produced embryos will be employed as a last resort in the event that conditions for fibroblasts or ES cells cannot be established. RELEVANT PUBLICATIONS Meng L, Wolf DP. Sperm-induced oocyte activation in the rhesus monkey nuclear and cytoplasmic changes following intracytoplasmic sperm injection. Hum Reprod 12:1062-1068, 1997. Meng L, Ely JJ, Stouffer RL, Wolf DP. Rhesus monkeys produced by nuclear transfer. Biol Reprod 57:454-459, 1997.
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