Conditional gene trapping

描述（由申请人提供）：基因捕获是一种随机 插入突变，允许可视化捕获的基因表达。 由于基因捕获可以在小鼠胚胎干（ES）细胞中进行， 在理论上可以创建并永久存储 代表小鼠基因组中每个基因的突变等位基因。ES细胞携带a 基因陷阱插入可用于产生相应的小鼠品系。 因此，这样的资源将是非常宝贵的探索表达和 哺乳动物基因的功能，单独和集体，在一个方便的 模型系统由于目前的证据表明，许多基因具有多重 除了通过评估无效等位基因而明显的作用外， 希望插入突变是有条件的。这样，null 基因型可以在动物体内的特定时间和地点诱导。的 用于鉴定捕获基因的标准方法是基于RNA的，利用 昂贵且技术上具有挑战性的RT-PCR技术。与imperial 完成小鼠基因组，应该可以使用更便宜和更简单的 基于DNA的技术，以指定基因陷阱插入的位置。另一 设计基因陷阱插入文库的重要考虑是 ES细胞系来源的小鼠品系。突变 应该在适合最广泛的遗传背景下产生， 一系列应用程序。目前，最广泛使用的ES细胞系是 来源于各种129衍生的菌株，这些菌株不适合于许多 问题研究该提案的具体目标是：1）产生和 测试多功能、条件性基因捕获载体，2）开发机器人， 鉴定基因陷阱插入的基于DNA的方法和3）产生和 表征新的ES细胞系，其遗传背景与广泛的 一系列的研究，包括听觉系统的研究。成功完成 这些目标将为大规模基因陷阱细胞系奠定基础 生产，这将反过来加速小鼠模型的生产， 人类遗传疾病

DESCRIPTION (provided by applicant): Gene trapping is a method of random insertional mutagenesis that permits visualization of trapped gene expression. Since gene trapping can be carried out in mouse embryonic stem (ES) cells, it is theoretically possible to create and permanently store a collection of mutant alleles representing every gene in the mouse genome. ES cells carrying a gene trap insertion can be used to generate the corresponding mouse strain. Thus, such a resource would be invaluable for exploring the expression and function of mammalian genes, individually and collectively, in a convenient model system. Since current evidence suggests that many genes have multiple roles, beyond the ones evident by assessing a null allele, it is highly desirable that the insertion mutations be conditional. In this way, the null genotype could be induced at specific times and places within the animal. The standard methods for identification of trapped genes are RNA-based, utilizing expensive and technically challenging RT-PCR techniques. With the immanent completion of the mouse genome it should be possible to use cheaper and simpler DNA-based techniques to specify the location of gene trap insertions. Another important consideration in the design of a library of gene trap insertions is the strain of mouse from which the ES cell line is derived. The mutations should be generated in a genetic background that is suitable for the widest array of applications. Currently, the most widely used ES cell lines are derived from various 129-derived strains, which are not appropriate for many studies. The specific aims of this proposal are designed to 1) generate and test multifunctional, conditional gene trap vectors, 2) develop robotic, DNA-based methods of identifying gene trap insertions and 3) generate and characterize new ES cell lines with genetic backgrounds compatible with a wide array of studies, including those of the auditory system. Successful completion of these aims will set the stage for large-scale gene trap cell line production, which will, in turn, accelerate the production of mouse models of human genetic disease.