Transposon-based Platform for Insertional Mutagenesis and Genome Engineering

基于转座子的插入突变和基因组工程平台

• 批准号: 7936658
• 负责人: Darius Balciunas
• 金额: $ 31.54万
• 依托单位: TEMPLE UNIV OF THE COMMONWEALTH
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-08-01 至 2015-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7936658
• 关键词: Biological Assay; Biological Models; Cells; Characteristics; Chromosomal Rearrangement; Chromosome Deletion; Chromosomes; Complement; Development; Diphtheria Toxin; Evaluation; Gene Expression; Generations; Genes; Genetic; Genetic Models; Genome engineering; Goals; Grant; Human; Insertional Mutagenesis; Libraries; Mutate; Other Genetics; Pattern; Phenotype; Plasmids; Play; Principal Investigator; Process; Proteins; Regulation; Reporter; Reporting; Research Personnel; Role; Site; System; Tamoxifen; Testing; Time; Tissues; Transgenic Organisms; Variant; Vertebral column; Zebrafish; abstracting; base; cost; endodeoxyribonuclease SceI; gene function; human disease; improved; interest; loss of function; promoter; public health relevance; recombinase; research study; tool; vector; zebrafish genome

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.

描述（由首席研究员提供）：基于转座子的插入突变和基因组工程平台 摘要：研究人类基因功能的最快、最全面和最具成本效益的方法是通过斑马鱼等模型系统中的对应物。 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，首次在脊椎动物中同时测定这两个特征。我们的载体将使用 Gal4/UAS 报告基因表达模式，同时有条件地突变斑马鱼的基因。 Gal4/UAS 将使研究人员能够使用我们的基因陷阱作为驱动线，在特定组织中表达其他感兴趣的蛋白质。基因捕获盒的两侧是位点特异性重组酶识别位点的组合，用于调节致突变性。在改进和测试我们的载体的过程中，我们将生成、传播并提供 150 个斑马鱼基因捕获系。 为了补充这些基因捕获系，我们将生产几个具有组织特异性表达 Cre 重组酶的系，用于诱变性的空间调节，并使用它们恢复与生存必需的基因的整合。这些组织特异性 Cre 系也可用于遗传谱系追踪。 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，因此 120 个基因陷阱系将构成分布在整个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使用位点特异性重组酶产生大倒位和缺失的原理实验证明，并使其他研究人员能够生成感兴趣的斑马鱼“设计染色体”。 公共卫生相关性：遗传学在许多人类疾病中发挥着重要作用。该项目将开发工具来大大增强我们研究脊椎动物模型系统——斑马鱼中基因功能的能力。