New Directions in Mutagenized ES Cell Libraries

诱变 ES 细胞文库的新方向

• 批准号: 6523510
• 负责人: ANDRAS NAGY
• 金额: $ 22.5万
• 依托单位: SINAI HEALTH SYSTEM
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-09-29 至 2004-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6523510
• 关键词: biotechnology; cell line; chromosome aberrations; electroporation; embryonic stem cell; expression cloning; functional /structural genomics; gene expression; genetic library; genetic manipulation; high throughput technology; information systems; laboratory mouse; molecular cloning; phenotype; polymerase chain reaction; proteomics; recombinase; sequence tagged sites; site directed mutagenesis; southern blotting; technology /technique development; transfection /expression vector

DESCRIPTION (provided by applicant): This project will develop new tools to facilitate high throughput, genome-wide mutagenesis in mouse embryonic Stem (ES) cells. Functional analysis of the mouse genome is a critical component of functional genomics, providing information relevant to human gene function and disease states. Gene trap mutagenesis in ES cells is a proven technology for generating sequence-tagged insertional mutations in mice. We propose to develop new gene trap vectors, incorporating a novel integrase-mediated recombination system. This vector will be used to generate a library of ES clones containing insertions within a wide range of genes across the genome. This library will be screened for expression via in vitro expression assays, using differentiation protocols already developed in the group. Each insertion will also have a RACE sequence tag associated with it and all information will be incorporated into an online database for availability to the community. Interesting insertions can be used to study mutant phenotype after germ line transmission, or can be used as sites of further gene modification or addition, by means of the integrase system. A complementary strategy of mutagenesis in ES cells will also be developed, focusing on in vitro recessive phenotypic screening, combining chromosome-specific loss of heterozygosity with chemical mutagenesis of ES cells. Both strategies will be developed first in existing 129 ES cells, but will be transferred to newly derived C57BL/6 cell lines, as soon as they are validated, to provide mutations on the B6 'gold standard' background.

描述（由申请人提供）：该项目将开发新的工具， 促进小鼠胚胎干细胞中的高通量、全基因组诱变 (ES)细胞小鼠基因组的功能分析是基因组学的重要组成部分。 功能基因组学，提供与人类基因功能相关的信息， 疾病状态。ES细胞中的基因陷阱诱变是一种经过验证的技术， 在小鼠中产生序列标记的插入突变。我们建议发展 新的基因诱捕载体，其结合了新的整合酶介导的重组 系统该载体将用于产生ES克隆文库，其含有： 在基因组中的大范围基因内的插入。这个图书馆将是 通过体外表达测定筛选表达，使用分化 已经在小组中制定的协议。每个插入也将有一个RACE 与之相关的序列标签，并且所有信息将被并入 一个在线数据库，供社区使用。有趣的插入 可用于研究种系传递后的突变表型，或者可 作为进一步基因修饰或添加的位点，通过 整合酶系统在ES细胞中进行诱变的补充策略也将 重点进行体外隐性表型筛选，结合 ES化学诱变染色体特异性杂合性丢失 细胞这两种策略将首先在现有的129个ES细胞中开发， 将被转移到新衍生的C57 BL/6细胞系中，一旦它们被 验证，以提供B6“金标准”背景上的突变。