Development of versatile gene vector designs

多功能基因载体设计的开发

• 批准号: 6447123
• 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/6447123
• 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来在小鼠中产生插入突变 可立即进行分子分析。但也存在一定的不足 该技术已得到认可，需要在开始之前解决 大规模努力捕获哺乳动物体内大约 30,000 个基因中的每一个 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） 目前正在进行努力。更重要的是，常规基因的多功能性 捕获方法是有限的，并且不允许生成其他、 更有用的是，完全解决基因功能所需的等位基因。目标 这笔赠款的目的是开发最佳的基因陷阱载体设计和方法 小鼠基因的大规模功能分析。具体来说，这 提案旨在分离具有高度种系能力、不依赖饲养者的 来自C57BL/6品系小鼠的胚胎干细胞，构建“万能” 基因陷阱载体能够针对所有类别的基因，并设计和 通过位点特异性重组测试基因捕获载体的可行性 网站。位点特异性重组位点的战略布局将允许 基因陷阱位点的二次修饰能够创建 无限多种理想的等位基因。这些修改包括但不限于 不限于，1) 等位基因突变系列的产生 捕获基因（无效、亚等位突变和位点特异性突变），2) 创造 条件等位基因（时间和组织特异性），3）包含 用于研究基因表达和细胞功能的报告基因（Bgal、 GFP、PLAP等）、4）重组酶的引入和控制表达 (Cre、fip等)和反式激活剂(TetR、gal4等)、5)的生产 异源基因产物的敲入，以及6)阴性药物的添加 用于工程染色体缺失的选择盒（HPRT、TK）和 倒置。本提案中描述的技术将具有广泛的应用前景 应用到哺乳动物生物学的所有领域，并将补充和扩展 小鼠的其他诱变策略。