SITE DIRECTED TRANSGENICS USING THE CRELOX SYSTEM

转基因动物是阐明正常基因表达的宝贵工具。 和不正常的生物过程。 因此， 被用来研究一系列与健康相关的过程。 虽然 转基因动物的生产有几种方法，大多数是 通过原核注射或同源重组产生， 胚胎干细胞（ES细胞）。虽然小鼠的原核技术 虽然发展得很好，但仍然需要相当多的技术专长， 时间和资源来通过同源重组生产小鼠。 作为 用传统方法生产转基因动物的替代方法 囊胚或原核注射方案，位点特异性 正在提出重组系统来指导转基因进入 预定的染色体位点。 一个特定地点的系统， 显示在哺乳动物细胞中由以下组成的Cre-loxP中有功能 重组酶（Cre）及其DNA识别位点（loxP）。 我们建议 开发一个基于Cre-loxP的系统，用于定点引进 转基因到哺乳动物基因组的预定位置。 这 该技术应适用于鼠和非鼠物种 并将广泛造福于生物医学界。 本申请的总体目标包括：1）识别 的突变loxP位点，有利于Cre介导的整合反应。 一 细菌诱变分析系统将用于确定 loxP位点的不同突变对 插入。2)测试突变型loxP位点的效率， 在aim 1中鉴定的插入反应，以及突变体 将在小鼠的WAP基因座中进行测试， 胚胎干细胞 将在WAP中引入突变loxP位点 基因座的同源重组和Cre介导的频率 整合决定。3)确定Cre介导的 通过原核注射进行整合。作为最终的目标， 应用是开发定点转基因系统， 可以通过原核注射使用，我们建议产生转基因 含有loxP标记的测试基因座的小鼠系。 这个转基因品系 将用于确定影响频率的因素， 当使用原核注射时，突变loxP位点的整合。这 建议是发展新的、可靠的 哺乳动物基因组定点修饰技术。

Transgenic animals are invaluable tools for the elucidation of normal and abnormal biological processes in intact systems. As such they have been used to study a host of health-related processes. Although there are several methods for the production of transgenic animals, most are generated by pronuclear injection or by homologous recombination in embryonic stem (ES) cells. While the pronuclear technology in mice is well developed, it still requires a considerable technical expertise, time, and resources to produce mice by homologous recombination. As an alternative to the production of transgenic animals by traditional blastocyst or pronuclear injections protocols, site-specific recombination systems are being proposed to direct transgenes into predetermined chromosomal sites. One site- specific system which has shown to be functional in mammalian cells in the Cre-loxP composed of a recombinase (Cre) and its DNA recognition site (loxP). We propose to develop a Cre-loxP based system for the site-directed introduction of transgenes into predetermined locations of the mammalian genome. This technology should be applicable to both murine, and non-murine species and would widely benefit the biomedical community. The overall objective of this application include: 1) The identification of mutant loxP sites that favor Cre-mediated integration reaction. A bacterial mutagenesis analysis system will be utilized to determine the effects of different mutations in the loxP sites on the frequency of insertion. 2) Testing the efficiency of the mutant loxP sites favoring the insertion reaction identified in aim 1, as well as mutants previously described by others, will be tested in the WAP locus of mouse embryonic stem cells. Mutant loxP sites will be introduced in the WAP locus by homologous recombination and the frequency of Cre-mediated integration determined. 3) Determining the frequency of Cre-mediated integration by pronuclear injection. As the eventual aim of this application is the development of a site-directed transgenic system that can be used by pronuclear injection we propose to generate a transgenic mouse line containing a loxP-tagged test locus. This transgenic line will be used to determine the factors affecting the frequency of integration of mutant loxP sites when using pronuclear injection. This proposal is the first step in the development of a new, reliable technique for the site-directed modification of the mammalian genome.