RETROTRANSPOSON INSERTIONAL MUTAGENESIS OF MOUSE

获取小鼠和人类的完整DNA序列的努力将产生关于哺乳动物基因组内容的大量信息。然而，最深刻的生物学洞察只会出现在序列与描述每个基因在有机体中的作用的联系上。在本申请中，我们描述了一种允许在小鼠基因组中产生随机插入突变的方法。在所提出的系统的设计中固有的是用于通过视觉检查来检测插入事件的存在的方法。事实上，毛色可以用来区分插入事件的动物是杂合子还是纯合子，这样就可以在不需要分子分析的情况下进行遗传杂交。我们已经使用了这种通用方法的前身来产生和分析大约250个随机突变的小鼠品系。在这些人中，大约10%的人表现出明显有趣的表型。有几个显示出颅面缺陷，如腭裂。然而，目前这种方法的设计有几个缺陷，最终导致克隆与观察到的表型有关的基因需要几年时间。在这项应用中，我们建议创建一个大大改进的系统版本，它结合了当前设计的成功方面，但提供了分离因插入而中断的基因的简化方法。这将通过使用重组逆转录病毒来实现，这些重组逆转录病毒被包装在睾丸的Sertoli细胞中，并作为新的整合位点仅通过雄性生殖系传播。这种方法最初将被应用于产生颅面部缺陷的小鼠模型；然而，如果成功，该方法也将得到更广泛的应用。拟议的商业应用：这项建议描述了一种方法学的创建，可用于创建小鼠随机插入突变的文库，并提供了为具有特定所需表型的小鼠筛选这些文库的方法。这样的库可以用来识别疾病的小鼠模型，这种模型的使用将在工业中广泛使用。

Efforts to obtain the complete DNA sequences of mouse and man will yield an enormous amount of information regarding the content of mammalian genomes. However, the most profound biological insights will come only upon linkage of sequences with a description of the role of each gene in the organism. In this application, we describe a methodology that permits the generation of random insertional mutations in the mouse genome. Inherent in the design of the proposed system are methods for detecting the presence of an insertion event by visual inspection. In fact, coat color can be used to distinguish whether animals are heterozygous or homozygous for the insertion event, allowing genetic crosses to be followed without the need for molecular analysis. We have used a previous incarnation of this general approach to generate and analyze approximately 250 strains of randomly mutated mice. Of these, approximately 10% showed overtly interesting phenotypes. Several displayed craniofacial defects such as cleft palate. However, the current design of the approach has several deficiencies that ultimately result in it taking several years to clone genes responsible for observed phenotypes. In this application, we propose to create a vastly improved version of the system that incorporates the successful aspects of the current design but that provides simplified methods for isolating the genes that have been disrupted by insertion. This will be accomplished through the use of recombinant retroviruses that are packaged in the sertoli cells of the testes and that are transmitted as new integration sites only through the male germ line. This methodology will initially be applied to the generation of mouse models with craniofacial defects; however, if successful, the approach will also find much broader application. PROPOSED COMMERCIAL APPLICATIONS: This proposal describes the creation of a methodology that can be used to create libraries of random insertional mutations in mouse and provides ways to screen those libraries for mice having specific desired phenotypes. Such a library could be used to identify mouse models of disease, the use of which would be widespread in industry.