Development of versatile gene vector designs

描述(申请者提供)：小鼠胚胎干细胞中的基因捕获 Cells提供了一个强大的工具来在小鼠身上产生插入突变 可以立即进行分子分析。然而，在某些方面存在缺陷 这项技术已经得到认可，需要在开始之前解决 一项大规模的努力，以捕获哺乳动物大约30,000个基因中的每一个 基因组。这些缺点包括所使用的小鼠品系的不相容 基因捕获(和基因打靶)与经典ENU突变的关系 目前正在进行的努力。更重要的是，传统基因的多功能性 诱捕方法是有限的，并且不允许产生其他， 更有用的是，完全解决基因功能所需的等位基因。目标是 这笔赠款的目的是开发最优的基因陷阱载体设计和方法 对小鼠基因的大规模功能分析。具体地说，这 一项旨在分离高度生殖系能力、不依赖饲养者的提案 C57BL/6系小鼠胚胎干细胞构建“通用型” 基因陷阱载体能够针对所有类别的基因，并设计和 用定点重组技术检测基因捕获载体的可行性 网站。战略性地安置特定地点的重组地点将允许 基因陷阱基因座上的二次修改使创建和 无限种类的理想等位基因。这些修改包括但不限于 不限于，1)产生一系列等位基因突变 陷阱基因(零突变、亚型突变和定点突变)，2)创造 条件等位基因(时间和组织特有的)，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)生产 异源基因产物的敲入；6)阴性药物的添加 选择盒(HPRT，TK)用于工程染色体删除和 反转。这项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 在老鼠身上的其他诱变策略。

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.