Conditional gene trapping

描述(申请人提供)：基因捕获是一种随机的方法 插入突变，使捕获的基因表达可视化。 由于基因捕获可以在小鼠胚胎干细胞中进行，因此它 在理论上可以创建并永久存储 代表老鼠基因组中每一个基因的突变等位基因。携带A基因的ES细胞 基因陷阱插入可以用来产生相应的小鼠品系。 因此，这样的资源对于探索表达式和 哺乳动物基因的功能，单独和集体，在一个方便的 模型系统。因为目前的证据表明许多基因有多个 角色，除了通过评估零等位基因而明显的角色之外，它是高度 希望插入突变是有条件的。通过这种方式，空值 在动物体内，可以在特定的时间和地点诱导出基因型。这个 识别捕获基因的标准方法是基于RNA的，利用 昂贵且具有技术挑战性的RT-PCR技术。与其内在的 小鼠基因组的完成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。另一个 在设计基因陷阱插入文库时的重要考虑因素是 小鼠的品系，ES细胞系就是从这个品系衍生出来的。突变 应该在适合最广泛的人的遗传背景下产生 一系列应用程序。目前，使用最广泛的ES细胞系是 派生自各种129派生的菌株，这些菌株不适合许多人 学习。本提案的具体目标是：1)产生和 测试多功能、有条件的基因捕获载体；2)开发机器人， 基于DNA的识别基因陷阱插入的方法和3)产生和 鉴定新的ES细胞系，其遗传背景与广泛的 一系列研究，包括那些关于听觉系统的研究。成功完成 这些目标将为大规模基因陷阱细胞系的建立奠定基础 生产，这将反过来加速鼠标模型的生产 人类遗传病。

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.