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: 8496840
• 负责人: Darius Balciunas
• 金额: $ 28.92万
• 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/8496840
• 关键词: Biological Assay; Biological Models; Cells; Characteristics; Chromosomal Rearrangement; Chromosome Deletion; Chromosome inversion; Chromosomes; Complement; Development; Diphtheria Toxin; Evaluation; Gene Expression; Gene Expression Profile; 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 effective; 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.

描述（由主要研究者提供）：基于转座的插入诱变和基因组工程摘要的平台摘要：研究人类基因功能的最快，最全面，最具成本效益的方式是通过其在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，这些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，同时在斑马鱼中有条件地突变基因。 GAL4/UAS将使研究人员能够将我们的基因陷阱用作驱动线，以表达特定组织中感兴趣的其他蛋白质。基因陷阱盒将以特异性重组酶识别位点的结合来调节诱变性。在改进和测试媒介的过程中，我们将生成，传播并提供150个斑马鱼基因陷阱线。 为了补充这些基因陷阱线，我们将生产几条CRE重组酶的组织特异性表达，以用于诱变的空间调节，并使用它们将其恢复为可生存力必不可少的基因。这些组织特异性的CRE系也可用于遗传谱系追踪。 由于我们的基因陷阱将包含用于特定位点特异性重组酶的识别位点，因此120个基因陷阱线将构成分布在整个斑马鱼基因组中的重组酶识别位点的库。这将使我们能够进行原理实验证明，用于使用特定地点的重组酶来产生大型反转和缺失，并使其他研究人员能够生成斑马鱼“设计师染色体”。