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: 7326500
• 负责人: JONATHAN M. AUERBACH
• 金额: $ 11.58万
• 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/7326500
• 关键词: Biological Assay; Cell Line; Cell Nucleus; Cell Therapy; 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; Modality; Monitor; Mus; National Center for Research Resources; Numbers; Pathway interactions; Phase; Play; Principal Investigator; Protocols documentation; Quality Control; Rate; Research; Research Personnel; Resources; Role; Safety; Screening procedure; Small Business Funding Mechanisms; Small Business Innovation Research Grant; Source; Standardization; Stem cells; Sterility; Study models; Terminal Repeat Sequences; Testing; Transgenes; Transgenic Mice; Translational Research; 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

DESCRIPTION (provided by applicant): 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. The ultimate goal of this application is to propagate and completely characterize two ES cell lines from useful mouse models and to make these ES lines readily accessible to the scientific community. We expect that this work will play a critical role in the scientific community and will become a valuable resource. The mouse ES cell lines will be available to all. The ES cells we will provide will be used as the starting point for the determination of gene function, as well as for in vitro elucidation of differentiation pathway mechanisms. As embryonic stem cells advance closer to becoming a source for cell-based therapies, these standardized cells and our program can form the nucleus of modalities for translational research.

描述（由申请方提供）：实验室小鼠是研究人类疾病的常用模型。近年来，小鼠也被广泛用于哺乳动物基因功能的分析。小鼠的基因组相对容易获得，因此许多转基因小鼠已经被工程化。小鼠胚胎干细胞中的同源重组已经成为创造这些转基因小鼠的便利载体。基因功能测定的一个主要限速因素是缺乏来自各种遗传背景的经验证的生殖系小鼠ES细胞系。NIH发起的名为“敲除小鼠计划（KOMP）”的新项目致力于在C57 B1/6遗传背景下产生小鼠基因组中基因的完整或接近完整的敲除突变。我们的建议是对KOMP项目的补充，因为我们建议使用的一条生产线是C57 Bl/6（生产线LB 10），另一条生产线是F1混合动力C57 Bl/6 x 129（生产线LC 3）。两个品系对于普遍表达的GFP转基因都是纯合的。为了能够比较不同实验室的结果，需要在相同的起始物料上进行研究。关于ES细胞系的繁殖，存在大量的基因型和表型变化的证据，部分是由于培养过程中的不当处理引起的。在这里，我们建议标准化，繁殖，并彻底表征两个小鼠ES细胞系，LC 3和LB 10。这项应用的最终目标是使科学界容易获得来自有用小鼠模型的ES细胞系。具体而言，我们的目标是：目标一。建立每个小鼠ES细胞系的主细胞库和工作细胞库。Aim II.对两种小鼠ES细胞系进行充分表征，并准备进行分销。目标I下的工作将包括两个细胞系培养条件的标准化和在这些条件下细胞的扩增，而目标II下，我们将监测体外繁殖过程中多能性的变化。我们对多能性的最终测试将是检测ES细胞系在小鼠生殖系中的繁殖能力。未来的工作将包括这些品系的规模扩大和分布到科学界，沿着将其他重要的小鼠ES品系纳入该计划。本申请的最终目标是从有用的小鼠模型中繁殖和完全表征两种ES细胞系，并使这些ES细胞系易于为科学界所用。我们希望这项工作将在科学界发挥关键作用，并成为宝贵的资源。小鼠ES细胞系将向所有人开放。我们将提供的ES细胞将被用作确定基因功能的起点，以及用于体外阐明分化途径机制。随着胚胎干细胞越来越接近成为基于细胞的疗法的来源，这些标准化的细胞和我们的计划可以形成转化研究模式的核心。