Development of versatile gene vector designs

多功能基因载体设计的开发

• 批准号: 6607445
• 负责人: William C. SKARNES
• 金额: $ 30.08万
• 依托单位: UNIVERSITY OF CALIFORNIA BERKELEY
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-09-19 至 2004-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6607445
• 关键词: alleles; biotechnology; cell line; embryonic stem cell; gene expression; gene mutation; gene targeting; genetic manipulation; genetic transcription; genetically modified animals; laboratory mouse; site directed mutagenesis; technology /technique development; tissue /cell culture; transfection; transfection /expression vector; transposon /insertion element

DESCRIPTION (provided by applicant): Gene trapping in mouse embryonic stem cells offers a powerful tool to create insertional mutations in mice that are immediately accessible to molecular analysis. However, certain deficiencies in the technology have been recognized and need to be addressed before embarking on a large-scale effort to trap each of the 30,000 or so genes in the mammalian genome. These drawbacks include the incompatibility of the mouse strains used for gene trapping (and gene targeting) in relation to classical ENU mutagenesis efforts now underway. More importantly, the versatility of conventional gene trapping approaches is limited and does not allow for the generation of other, more useful, alleles that are needed to fully address gene function. The goal of this grant is to develop optimal gene trap vector designs and methods for the large-scale functional analysis of genes in mice. Specifically, this proposal aims to isolate highly germline-competent, feeder-independent embryonic stem cells from the C57BL/6 strain of mice, to construct "universal" gene trap vectors capable of targeting all classes of genes, and to design and test the feasibility of gene trap vectors with site-specific recombination sites. The strategic placement of site-specific recombination sites will permit secondary modifications at the gene trap locus enabling the creation an unlimited variety of desirable alleles. These modifications include, but are not limited to, 1) the generation of an allelic series of mutations in the trapped gene (null, hypomorphic, and site-specific mutations), 2) the creation of conditional alleles (temporal and tissue-specific), 3) the inclusion of reporter genes for the study of gene expression and cellular functions (Bgal, GFP, PLAP, etc.), 4) the introduction and controlled expression of recombinases (Cre, fip, etc.) and transactivators (TetR, gal4, etc.), 5) the production of knock-ins of heterologous gene products, and 6) the addition of negative drug selection cassettes (HPRT, TK) for engineering chromosomal deletions and inversions. The technology described in this proposal will have wide applications to all areas of mammalian biology and will complement and extend other mutagenesis strategies in mice.

描述（由申请人提供）：小鼠胚胎干中的基因捕获 细胞提供了一个强大的工具，在小鼠中产生插入突变， 立即进行分子分析。然而，某些缺陷， 这些技术已经得到承认，需要在开始之前加以解决 在大规模的努力，以捕捉每一个30,000左右的基因在哺乳动物 基因组这些缺点包括所用小鼠品系的不相容性 用于与经典ENU诱变相关的基因捕获（和基因靶向） 目前正在努力。更重要的是，常规基因的多功能性 捕获方法是有限的并且不允许产生其它的， 更有用的等位基因，这些等位基因是完全解决基因功能所必需的。目标 这项拨款的目的是开发最佳的基因陷阱载体设计和方法， 大规模的小鼠基因功能分析。具体来说， 该提案旨在分离高度种系能力，饲养者独立的 胚胎干细胞从C57 BL/6小鼠品系，以构建“通用” 能够靶向所有类型基因的基因捕获载体，并设计和 测试具有位点特异性重组的基因诱捕载体的可行性 网站.位点特异性重组位点的策略性放置将允许 在基因陷阱位点的二级修饰使得能够产生 无限多样的理想等位基因。这些修改包括但 不限于，1）在基因组中产生等位基因系列突变， 被困基因（无效，亚纯型和位点特异性突变），2）创建 条件等位基因（时间和组织特异性），3）包括 用于研究基因表达和细胞功能的报道基因（Bgal， GFP、PLAP等），4)重组酶的引入和受控表达 (Cre、fip等）和反式激活因子（TetR、gal 4等），5)生产 外源基因产物的敲入，以及6）阴性药物的添加 - 用于工程化染色体缺失的选择盒（HPRT，TK），以及 倒置。本提案中描述的技术将具有广泛的 应用于哺乳动物生物学的所有领域，并将补充和扩展 其他诱变策略。