RETROTRANSPOSON INSERTIONAL MUTAGENESIS OF MOUSE

获得小鼠和人的完整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，最深刻的生物学见解将只出现在序列的链接与每个基因在生物体中的作用的描述。在这个应用中，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。所提出的系统的设计中固有的是用于通过目视检查检测插入事件的存在的方法。事实上，毛色可以通过插入事件来区分动物是杂合子还是纯合子，从而可以在不需要分子分析的情况下进行遗传杂交。我们已经使用这种一般方法的前一个化身来生成和分析大约250株随机突变的小鼠。其中，大约10%的人表现出明显有趣的表型。几个表现出颅面缺陷，如腭裂。然而，目前这种方法的设计有几个缺陷，最终导致它需要几年的时间来克隆负责观察到的表型的基因。在这个应用程序中，我们建议创建一个系统的巨大改进版本，它结合了当前设计的成功方面，但提供了分离被插入破坏的基因的简化方法。这将通过使用重组逆转录病毒来实现，重组逆转录病毒包装在睾丸的支持细胞中，并且只能通过雄性生殖系作为新的整合位点传播。该方法将首先应用于颅面缺损小鼠模型的生成；然而，如果成功，这种方法还将得到更广泛的应用。拟议的商业应用：该提案描述了一种方法的创建，该方法可用于创建小鼠随机插入突变文库，并提供了筛选具有特定所需表型的小鼠文库的方法。这样的一个库可以用来识别疾病的小鼠模型，在工业上的应用将是广泛的。

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.