Oct4 and the Maintenance of Mammalian Stem Cells

Oct4 和哺乳动物干细胞的维持

• 批准号: 6766666
• 负责人: JAMES S KEHLER
• 金额: $ 8.57万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-09-17 至 2009-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6766666
• 关键词: RNA interference; biochemical evolution; cats; cell biology; cell differentiation; cell line; developmental genetics; embryogenesis; gene expression; genetic models; genetically modified animals; germ cells; mammalian embryology; model design /development; molecular biology; protein structure function; stem cells; transcription factor; transfection; transfection /expression vector

DESCRIPTION (provided by applicant): This proposal describes a 5-year training program for the development of the principal investigator into an independent investigator in the field of comparative medical genetics. James Kehler received his VMD in 2002, and is working on his Ph.D. thesis in Cell and Molecular Biology at the University of Pennsylvania. During the first 3 years of the proposal, the principal investigator will receive superb training in molecular and stem cell biology in the laboratory of Hans Scholer, his thesis advisor and sponsor of this proposal, at the Center For Animal Transgenesis and Germ Cell Research. He will have the opportunity to learn new transgenic techniques in mice and large animal species from additional researchers at this unique center. During the last 2 years of the training grant, the principal investigator will work in the laboratory of Dr. Stephen O'Brien, co-sponsor of this proposal, at the National Cancer Institute. Dr. O'Brien's Laboratory of Genomic Diversity focuses on the coevolution of mammals and retroviruses. Here the principal investigator will continue to develop his skills in molecular biology and reproductive medicine, while learning additional analytic techniques in genomics. The suitability of these research environments and the principal investigator's prior experience makes it feasible to propose to develop new transgenic cat models and stem cell lines to test the specific hypothesis of this proposal: The roles of Oct4 in maintaining pluripotency in Embryonic Stem Cells (ES cells) and the survival of Primordial Germ Cells (PGCs) are conserved in mammals. In Aim 1, transgenic Oct4-reporter cat embryos will be produced by pronuclear injection to help determine whether Oct4 expression is restricted to pluripotent cells and germ cells in the early embryo and fetus of the cat as in the mouse. In Aim 2, RNA interference will be used to determine if Oct4 is required for the maintenance of pluripotency in cat embryos and feline ES cells. In Aim 3, vector mediated transgenesis will be used to test whether Oct4 is required for the survival of feline PGCs. In addition, the potential feline ES and EG cells established in this proposal could be used for future gene-targeting experiments. One of the principal investigator's long-term goals is to realize the potential of the cat as a model species by developing the necessary resources for the research community. By the end of this training program, he will be thoroughly prepared to develop new animal models of human diseases and stem cell and gene therapies.

描述（由申请人提供）： 该提案描述了一项为期 5 年的培训计划，旨在将主要研究者发展为比较医学遗传学领域的独立研究者。 James Kehler 于 2002 年获得 VMD，目前正在攻读博士学位。宾夕法尼亚大学细胞和分子生物学论文。在该提案的前 3 年，首席研究员将在动物转基因和生殖细胞研究中心的论文导师和该提案的发起人 Hans Scholer 的实验室接受分子和干细胞生物学方面的一流培训。他将有机会向这个独特中心的其他研究人员学习小鼠和大型动物物种的新转基因技术。在培训资助的最后两年内，首席研究员将在国家癌症研究所的斯蒂芬·奥布莱恩博士（该提案的共同发起人）的实验室工作。奥布莱恩博士的基因组多样性实验室专注于哺乳动物和逆转录病毒的共同进化。在这里，首席研究员将继续发展他在分子生物学和生殖医学方面的技能，同时学习基因组学方面的其他分析技术。这些研究环境的适用性以及主要研究者之前的经验使得提议开发新的转基因猫模型和干细胞系来测试该提议的具体假设是可行的：Oct4在维持胚胎干细胞（ES细胞）多能性中的作用）和原始生殖细胞（PGC）的存活在哺乳动物中是保守的。在目标 1 中，将通过原核注射产生转基因 Oct4 报告基因猫胚胎，以帮助确定 Oct4 表达是否像小鼠一样仅限于猫早期胚胎和胎儿中的多能细胞和生殖细胞。在目标 2 中，将使用 RNA 干扰来确定 Oct4 是否是维持猫胚胎和猫 ES 细胞多能性所必需的。在目标 3 中，将使用载体介导的转基因来测试 Oct4 是否是猫 PGC 生存所必需的。此外，该提案中建立的潜在猫科动物 ES 和 EG 细胞可用于未来的基因靶向实验。首席研究员的长期目标之一是通过为研究界开发必要的资源来实现猫作为模式物种的潜力。培训计划结束时，他将为开发人类疾病以及干细胞和基因疗法的新动物模型做好充分准备。