Propogation and Characterization of 2 Mouse Embryonic Stem Cell Lines

2 种小鼠胚胎干细胞系的增殖和表征

• 批准号: 7497843
• 负责人: JONATHAN M. AUERBACH
• 金额: $ 2.89万
• 依托单位: GLOBALSTEM, INC.
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-08-16 至 2010-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7497843
• 关键词: Biological Assay; Cell Line; Cells; Communities; Condition; Data; ES Cell Line; Engineering; Future; Gene Mutation; Genetic; Germ Lines; Goals; Grant; Green Fluorescent Proteins; Hand; Hybrids; In Vitro; Knock-out; Knockout Mice; Laboratories; Laboratory mice; Monitor; Mus; National Center for Research Resources; Numbers; Phase; Principal Investigator; Protocols documentation; Quality Control; Rate; Research; Research Personnel; Resources; Safety; Screening procedure; Small Business Funding Mechanisms; Small Business Innovation Research Grant; Standardization; Stem cells; Sterility; Study models; Terminal Repeat Sequences; Testing; Transgenes; Transgenic Mice; Undifferentiated; Work; cell bank; embryonic stem cell; experience; gene function; homologous recombination; human disease; in vivo; mouse genome; mouse model; pathogen; pluripotency; programs; research study; scale up; transmission process

The laboratory mouse is a popular model for the study of human disease. In the recent past, the mouse has also become widely used for the analysis of mammalian gene function. The genome of the mouse is relatively accessible and, thus, many transgenic mice have been engineered. Homologous recombination in mouse embryonic stem cells has been a convenient vehicle for the creation of these transgenic mice. One major, rate-limiting factor for the determination of gene function has been the lack of access to validated, germline competent mouse ES cell lines from various genetic backgrounds. The newly launched NIH-sponsored program entitled Knockout Mouse Project (KOMP) is striving to generate a complete or nearly complete set of knockout mutations of the genes in the mouse genome on the C57Bl/6 genetic background. Our proposal is complementary to the KOMP project in that one of the lines we are proposing to use is a C57Bl/6 (line LB10) and the other line is an F1 hybrid C57Bl/6 x 129 (line LC3). Both lines are homozygous for a ubiquitously expressed GFP transgene. The ability to compare results across laboratories requires that studies be performed on the same starting material. Ample evidence for genotypic and phenotypic changes, partially induced through improper handling during culture, exists regarding the propagation of ES cell lines. Here, we propose to standardize, propagate, and thoroughly characterize two mouse ES cell lines, LC3 and LB10. The ultimate goal of this application is to make ES cell lines from useful mouse models readily accessible to the scientific community. Specifically, our aims are: Aim I. Establish Master and Working Cell Banks for each mouse ES cell line. Aim II. Fully characterize the two mouse ES cell lines and prepare them for distribution. Work under Aim I will include standardization of culture conditions for the two lines and expansion of the cells under those conditions, while under Aim II we will monitor for changes in pluripotency during in vitro propagation. Our ultimate test for pluripotency will be assaying the ES cell lines for their ability to populate the germ lines of mice. Future work will include scale-up and distribution of these lines to the scientific community along with the inclusion of other important mouse ES lines in this program.

实验室小鼠是研究人类疾病的流行模型。近 过去，小鼠也被广泛用于分析哺乳动物的基因 功能小鼠的基因组相对容易获得，因此，许多转基因小鼠都可以获得。 老鼠已经被改造过了。小鼠胚胎干细胞的同源重组 已经成为创造这些转基因小鼠的便利载体。一个少校， 决定基因功能的一个限速因素是缺乏获得 来自各种遗传背景的经验证的生殖系感受态小鼠ES细胞系。 新推出的NIH赞助的项目， 基因敲除小鼠项目（KOMP） 正在努力产生一个完整的或几乎完整的基因敲除突变的集合， 小鼠基因组中的C57 B1/6基因背景。我们的建议是 补充KOMP项目，我们建议使用的线路之一是 C57 B1/6（品系LB 10），另一个品系是F1杂种C57 B1/6 × 129（品系LC 3）。两 品系对于普遍表达的GFP转基因是纯合的。的能力 比较实验室之间的结果要求在相同的实验室中进行研究。 起始材料充分的证据表明基因型和表型的变化，部分 在培养过程中，由于处理不当而引起的， ES细胞系。在这里，我们建议标准化，宣传和彻底表征 两种小鼠ES细胞系，LC 3和LB 10。该应用程序的最终目标是使 ES细胞系来自科学界容易获得的有用的小鼠模型。 具体而言，我们的目标是： 艾姆岛建立每个小鼠ES细胞系的主细胞库和工作细胞库。 Aim II.充分表征两种小鼠ES细胞系，并将其制备用于 分布 目标I下的工作将包括两条生产线培养条件的标准化， 在这些条件下，细胞的扩增，而在目标II下，我们将监测 在体外繁殖过程中多能性的变化。我们对多能性的终极测试 检测胚胎干细胞系在小鼠生殖细胞系中的繁殖能力。未来 工作将包括扩大这些生产线的规模，并将其分发给沿着的科学界 在这个程序中包括其他重要的小鼠ES系。