Transposon-based Platform for Insertional Mutagenesis and Genome Engineering

摘要：研究人类基因功能最快、最全面、最具成本效益的方法是通过斑马鱼等模型系统中的对偶体。任何基因的两个关键特征是表达模式和功能表型的丧失。我们建议开发工具，首次在脊椎动物同时分析这两个特征。我们的载体将在斑马鱼中使用Gal4/UAS报告基因表达模式，同时对基因进行条件突变。Gal4/UAS将使研究人员能够使用我们的基因陷阱作为驱动系，在特定组织中表达其他感兴趣的蛋白质。基因诱捕盒的两侧是位点特异性重组酶识别位点的组合，用于调控诱变性。在改进和测试我们的载体的过程中，我们将产生、繁殖和提供150个斑马鱼基因诱捕系。为了补充这些基因诱捕系，我们将生产几种具有组织特异性表达Cre重组酶的系，用于空间诱变性调控，并利用它们将整合恢复为生存所必需的基因。这些组织特异性Cre系也将用于遗传谱系追踪。由于我们的基因陷阱将包含位点特异性重组酶的识别位点，120个基因陷阱系将构成一个分布在斑马鱼基因组中的重组酶识别位点文库。这将使我们能够进行原理验证实验，使用位点特异性重组酶来产生大的反转和缺失，并使其他研究人员能够产生感兴趣的斑马鱼“设计染色体”。

DESCRIPTION (provided by Principal Investigator): Transposon-Based Platform for Insertional Mutagenesis and Genome Engineering Abstract: The fastest, most comprehensive and cost-effective way to study human gene function is through their counterparts in model systems such as zebrafish. Two key characteristics of any gene are expression pattern and loss of function phenotype. We propose to develop tools that for the first time in a vertebrate simultaneously assay both of these characteristics. Our vectors will report gene expression patterns using Gal4/UAS while conditionally mutating genes in zebrafish. Gal4/UAS will enable investigators to use our gene traps as driver lines to express other proteins of interest in specific tissues. Gene trap cassettes will be flanked by a combination of site-specific recombinase recognition sites for regulation of mutagenicity. During the process of improving and testing our vectors we will generate, propagate and make available 150 zebrafish gene trap lines. To complement these gene trap lines, we will produce several lines with tissue-specific expression of Cre recombinase for spatial regulation of mutagenicity and use them to revert integrations into genes essential for viability. These tissue-specific Cre lines will be also useful for genetic lineage tracing. Since our gene traps will contain recognition sites for site-specific recombinases, the 120 gene trap lines will constitute a library of recombinase recognition sites distributed throughout the zebrafish genome. This will allow us to carry out proof of principle experiments for use of site-specific recombinases to generate large inversions and deletions, and enable other researchers to generate zebrafish "designer chromosomes" of interest. PUBLIC HEALTH RELEVANCE: Genetics plays a substantial role in many human diseases. This project will develop tools to greatly enhance our ability to study gene function in a vertebrate model system- the zebrafish.