FULL TERM DEVELOPMENT OF RHESUS MONKEYS PRODUCED BY NUCLEAR TRANSFER

基因相同的非人类灵长类动物可以提供强大的

Genetically identical nonhuman primates can provide a powerful animal model for gene therapy and research activities where the physiological parameters directly or indirectly under study are heritable. Here we demonstrate that nuclear transfer is a viable technology for the production of rhesus macaques. Mature (metaphase II) oocytes recovered from recombinant gonadotropin (Ares Serono)-treated females were enucleated by aspiration of the first polar body and underlying ooplasm. Then individual diploid blastomeres, recovered from in vitro fertilization (IVF)-produced embryos (either fresh or frozen-thawed) and used as nuclear donors, were injected under the zona pellucida of enucleated (chromosome-free) oocytes. Reconstituted oocyte-blastomere pairs were exposed to KSOM containing cycloheximide (7.5 fg/ml) and 10% neonatal calf serum (NCS) for 1 hr to induce ooplast activation, then placed individually in an electrical field. Membrane fusion was induced by an electric pulse (2 continuous square wave DC pulses of 2.2kv/cm for 50 f sec in 0.3 M mannitol, 100 fM CaCI2, 100 fMgSO4 and 0.5 mg PVP/ml) following alignment of the membranes to be fused, parallel to the DC electrodes. Oocyte-blastomere pairs received a second and third pulse at hourly intervals. Between pulses, the pairs were held in KSOM containing cycloheximide and cytochalasin B. Immediately after the third electric pulse, seventy-eight reconstituted embryos were co-cultured on buffalo rat liver (BRL) cells in CMRL-1066 medium+10% NCS, then cryostored until transfer to synchronized host mothers. Of the 9 females receiving a total of 29 reconstituted embryos, 3 became pregnant, resulting in two live births; one male and one female. The parentage of both offsprings resulting from nuclear transfer technology was established unequivocally by genetic typing with eight PCR-amplified highly variable DNA short tandem repeat (STR) loci. This provides the first demonstration of development to term of primate embryos reconstituted by nuclear transfer technology.