PRODUCTION OF CLONALLY DERIVED RHESUS MONKEYS

克隆恒河猴的生产

• 批准号: 6592335
• 负责人: DON P WOLF
• 金额: $ 11.11万
• 依托单位: OREGON HEALTH & SCIENCE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-05-01 至 2003-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6592335
• 关键词: Mammalia; animal tissue; biotechnology; genetic manipulation; genetically modified animals; growth /development; reproductive system; technology /technique

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.

通过核技术生产基因相同的动物 转移对生物医学研究很重要，因为它消除了 基因型变异允许用更少的数据获得更高的统计有效性 实验动物。 此外，基础调查 免疫系统的本质将变得可行。 我们成立了 成功的恒河猴体外受精（IVF）计划 俄勒冈地区灵长类研究中心曾是猴子的栖息地 采用去核卵母细胞核移植技术产生 作为 IVF 产生的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 改进恒河猴的核移植程序 确定可用于支持的供体核的来源 创建大克隆尺寸。 这是两个具体目标 (1) 优化供体核源以生产 通过核移植克隆衍生的恒河猴。 三种可能 将评估供体核的来源以进行常规生产 基因相同的动物，即来自个体的卵裂球 IVF 产生的胚胎（我们经过验证的方法）、原代培养物 成纤维细胞和永生化胚胎干（ES）细胞。 最初， 来自胎儿和成人成纤维细胞和 ES 细胞的细胞核，转染 绿色荧光蛋白可确认供体核 参与发展，将审查他们的能力 支持核移植后的植入前发育 中期 II 细胞质。 实验将确定细胞是否 周期分期对于细胞核成功重编程至关重要 转移后。 (2) 产生克隆衍生的恒河猴。 目标 1 下建立的供体核的最佳来源将是 用于生产克隆衍生的、转基因的 猴子。 将创建 50 个重组胚胎的克隆 成纤维细胞和ES细胞并冷冻保存。 转让涉及 同一克隆的2个胚胎将在同步宿主中进行 妈妈们。 IVF产生的胚胎将作为最后的手段 成纤维细胞或胚胎干细胞的条件不能满足的事件 已确立的。 相关出版物Meng L，Wolf DP。 精子诱导 恒河猴卵母细胞激活核和细胞质变化 胞浆内单精子注射后。 嗡嗡声再现 12：1062-1068， 1997.孟L，伊利JJ，斯托弗RL，沃尔夫DP。 恒河猴产生 通过核转移。 生物复制 57：454-459，1997。