PRODUCTION OF CLONALLY DERIVED RHESUS MONKEYS

通过核技术生产基因完全相同的动物 转让对生物医学研究具有重要意义，因为它消除了 基因型变异允许更大的统计有效性， 实验动物 此外，基本调查 免疫系统的本质将变得可行。 我们建立 一个成功的恒河猴体外受精（IVF）计划， 俄勒冈州地区灵长类动物研究中心，那里的猴子 通过使用去核卵母细胞的核移植技术产生的 作为受体细胞质和单个卵裂球， 胚胎作为核供体。 目前的实验旨在 通过以下方法改进恒河猴的核移植程序： 识别可用于支持所述供体核的供体核源， 创建大型克隆。 这是两个具体目标：（1） 优化供体核源， 克隆来源的恒河猴通过核移植。 三种可能 将对常规生产中的供体细胞核来源进行评价 基因完全相同的动物，即来自 体外受精产生的胚胎（我们证明的方法）， 成纤维细胞和永生化胚胎干（ES）细胞。 起初， 来自胎儿和成人成纤维细胞和ES细胞的细胞核，用 绿色荧光蛋白，用于确认供体细胞核 参与开发的能力将受到审查， 支持植入前发育， 中期II细胞质。 实验将确定细胞是否 周期分期对于细胞核的成功重编程至关重要 转移后。 (2)克隆恒河猴。 根据目标1建立的供体核的最佳来源将 用于生产克隆衍生的转基因 猴子 50个重组胚胎的克隆将被创建， 成纤维细胞和ES细胞并冷冻储存。 涉及以下方面的转让 同一克隆的2个胚胎将在同步宿主中进行 妈妈们 体外受精产生的胚胎将作为最后的手段， 成纤维细胞或ES细胞的条件不能 确立了习 相关出版物Meng L，Wolf DP. 精子诱导的 恒河猴卵母细胞激活过程中核质的变化 卵胞浆内单精子注射 Reprod 12：1062-1068， 1997. 孟L，伊利JJ，斯托弗RL，沃尔夫DP. 恒河猴生产的 通过核移植。 Biol Reprod 57：454-459，1997.

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.