NUCLEAR TRANSFER IN 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非常重要，因为它 消除了实验操作过程中的基因型变异， 用更少的动物实现更大的统计有效性。 我们 成功建立了体外受精（IVF）计划， 俄勒冈州地区灵长类动物研究中心，那里的恒河猴一直在 这是第一次通过核移植产生。 技术 使用去核的中期II卵母细胞作为受体胞质体（MII 细胞质）和来自IVF产生的胚胎的单个卵裂球， 核捐赠者 正在进行的研究旨在改善核 在恒河猴中的转移过程， 生物医学界可以利用的基因相同的动物 用于实验，同时评估效率和 这种不断发展的方法的成本效益。 三个具体目标 为1. 为了优化制备、熟化、活化和低 MII细胞质的温度储存。 条件 将建立MII细胞质的冷冻保存，并在体外 将寻求成熟条件以利用 潜在的大量卵巢卵母细胞; 2. 建立 全能源（分化为所有细胞类型的潜力 存在于成体中）胚胎细胞或培养的胚胎干细胞 (ES)作为核捐赠者。 干细胞系将从体内产生 产生胚泡并用分子标记稳定转染， 例如绿色荧光蛋白。 这些品系的全能性 将在核移植后的重构胚胎中进行评价。 此外，来自IVF产生的胚胎的内细胞团细胞将被 评估为核供体和ES细胞系的来源;和3. 通过常规或连续核移植生产恒河猴 并评估卵裂球分离和培养作为替代方案 克隆的方法。 我们将生产5套基因完全相同的 动物在最初的一年和更多的此后通过核移植。 此外，在早期分离IVF产生的胚胎的简单性 单个卵裂球或卵裂球群转移的卵裂阶段 将卵裂球移植到卵带上， 克隆将被评估。 相关出版物Meng L，伊利JJ， Stouffer RL，Wolf DP. 核移植产生的恒河猴。 Biol Reprod 57：454-459，1997. Meng L，Wolf DP. 精子诱导卵母细胞 在恒河猴细胞核和细胞质的变化激活 卵胞浆内单精子注射 Reprod 12：1062-1068， 1997.

The production of genetically-identical animals by nuclear transfer is of great importance to biomedical research, as it eliminates genotypic variation during experimental manipulation and allows greater statistical validity with fewer animals. We established a successful in vitro fertilization (IVF) program at the Oregon Regional Primate Research Center where rhesus monkeys have been produced for the first time by nuclear transfer. The technology employs enucleated, metaphase II oocytes as recipient cytoplasts (MII cytoplasts) and individual blastomeres from IVF-produced embryos as nuclear donors. Ongoing research is designed to improve the nuclear transfer procedure in rhesus macaques with the goal of making genetically-identical animals available to the biomedical community for experimental use while evaluating the efficiency and cost-effectiveness of this evolving approach. The three specific aims are 1. To optimize the preparation, maturation, activation and low temperature storage of MII cytoplasts. Conditions for the cryopreservation of MII cytoplasts will be established and in vitro maturation conditions will be sought to take advantage of the potentially large numbers of ovarian oocytes; 2. To establish a source of totipotent (potential to differentiate into all cell types present in the adult) embryonic cells or cultured embryonic stem cells (ES) as nuclear donors. Stem cell lines will be created from in vivo produced blastocysts and stably transfected with a molecular marker, such as green fluorescent protein. The totipotency of these lines will be evaluated in reconstituted embryos following nuclear transfer. Additionally, inner cell mass cells from IVF-produced embryos will be evaluated as nuclear donors and as a source of ES cell lines; and 3. To produce rhesus monkeys by conventional or serial nuclear transfer and evaluate blastomere separation and culture as an alternative method of cloning. We will produce 5 sets of genetically identical animals in the initial year and more thereafter by nuclear transfer. Also, the simplicity of separating IVF-produced embryos at early cleavage stages with the transfer of individual blastomeres or groups of blastomeres to surrogate zonae offers a low technology method of cloning that will be evaluated. RELEVANT PUBLICATIONS Meng L, Ely JJ, Stouffer RL, Wolf DP. Rhesus monkeys produced by nuclear transfer. Biol Reprod 57:454-459, 1997. 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.