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一个强大的 用于基因治疗和研究活动的动物模型， 直接或间接研究的生理参数是 遗传的 在这里，我们证明了核移植是一种可行的方法， 恒河猴的生产技术。 成熟（中期 II）从重组促性腺激素（战神）回收的卵母细胞 Serono）处理的雌性动物通过抽吸第一个 极体和下面的卵质。 然后是个体二倍体 卵裂球，从体外受精（IVF）产生的 胚胎（新鲜的或冻融的）并用作核供体， 注射到去核（无染色体）的透明膜下， 卵母细胞 将重构的卵母细胞-卵裂球对暴露于KSOM 含放线菌酮（7.5 fg/ml）和10%新生牛血清（NCS） 1小时以诱导卵质体活化，然后单独放置在 电场 电脉冲（2 连续方波直流脉冲，2.2 Ω/cm，50 f sec，0.3 M 甘露醇、100 μ M CaCl 2、100 μ M MgSO 4和0.5 mg PVP/ml）， 将待熔合的膜平行于DC电极对齐。 卵母细胞-卵裂球对每小时接受第二次和第三次脉冲 的间隔 在脉冲之间，这些对被保持在KSOM中， 放线菌酮和细胞松弛素B。在第三次电 将78枚重组胚与布法罗共培养 将大鼠肝（BRL）细胞置于CMRL-1066培养基+10%NCS中，然后冷冻保存 直到转移到同步的宿主母亲那里 在9名女性中， 接受了总共29个重组胚胎，3个怀孕， 导致两个活产婴儿;一男一女。 亲子关系 这两个后代的核移植技术， 通过基因分型明确建立了8个PCR扩增的 高度可变的DNA短串联重复序列（STR）基因座。 这提供了以下 灵长类动物胚胎发育到足月的第一次证明 通过核移植技术重组。

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.