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只有在序列与生物体中每个基因的作用描述的联系中才会出现。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，毛色可以用来区分动物是杂合还是纯合的插入事件，允许遗传杂交，而不需要分子分析。我们已经使用这种通用方法的前身来生成和分析大约250种随机突变小鼠。其中，约10%显示出明显有趣的表型。几个显示颅面缺陷，如腭裂。然而，目前的设计方法有几个缺陷，最终导致它需要几年的克隆基因负责观察到的表型。在本申请中，我们建议创建一个大大改进的系统版本，该系统结合了当前设计的成功方面，但提供了简化的方法来分离因插入而被破坏的基因。这将通过使用重组逆转录病毒来实现，所述重组逆转录病毒包装在睾丸的支持细胞中，并且仅通过雄性生殖系作为新的整合位点传播。这种方法最初将被应用于生成具有颅面缺陷的小鼠模型;然而，如果成功的话，这种方法也将得到更广泛的应用。拟议的商业应用：该提案描述了可用于在小鼠中创建随机插入突变文库的方法的创建，并提供了筛选具有特定所需表型的小鼠的那些文库的方法。 这样的库可用于识别疾病的小鼠模型，其用途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.