Genomic Engineering in Drosophila

描述(由申请人提供)：在这项提案中，我们描述了一种名为“基因组工程”的新型遗传操作系统的开发，该系统将允许对果蝇中选定的基因组位点进行有效、无限制和定向的修改。基因组工程是一个分两步走的过程。首先，通过优化的基因打靶程序，通过删除目的基因并将其替换为噬菌体整合酶FC31的小DNA重组位点来产生创建者敲除系。其次，利用FC31进行DNA整合，将修饰后的目的基因DNA重新导入到方正基因敲除系的原生基因中。由于FC31介导的DNA整合是高效和非歧视的，几乎任何所需的遗传修饰都可以以高通量效率产生。基因组工程作为果蝇基因组定向工程的工具，将产生深远而革命性的影响。首先，基因组工程克服了同源重组固有的低效和局限性。它提供了一种几乎不受限制的方法来产生目标基因的任何所需突变等位基因，用于遗传、生化和细胞生物学分析。其次，为了构建有用和信息丰富的人类疾病模型，基因组工程使将人类遗传损伤精确复制到果蝇的同源基因中成为可能。最后，从长远来看，通过系统地为保守和必要的基因生成创始人敲除系，我们将把果蝇转变为一种更高效、更多功能的遗传模式生物，它也将更容易被非苍蝇研究人员访问和吸引，以解决他们的生物学问题。公共卫生相关性：果蝇是解决人类疾病中关键生物学问题的领先遗传模型系统。基因组工程是一种新的强大的遗传工具，它将极大地促进分析复杂的疾病途径和构建更好的果蝇疾病模型。

DESCRIPTION (provided by applicant): In this proposal we describe the development of a novel genetic manipulation system termed "genomic engineering" that will permit efficient, unlimited and directed modification of a chosen genomic locus in Drosophila. Genomic engineering is a two-step process. First, through an optimized gene targeting routine, a founder knock-out line is generated by deleting the target gene and replacing it with a small DNA recombination site of phage integrase FC31. Second, DNA integration by FC31 is used to reintroduce modified target gene DNA into the native locus in the founder knock-out line. Since FC31 mediated DNA integration is highly efficient and non-discriminating, virtually any desired genetic modification can be generated with high throughput efficiency. Genomic engineering will have a profound and revolutionary impact as a tool for directed engineering of the Drosophila genome. First, genomic engineering overcomes the inherent inefficiency and limitations of homologous recombination. It provides a virtually unlimited approach to generate any desired mutant allele of a target gene for genetic, biochemical and cell biologic assays. Second, for constructing useful and informative human disease models, genomic engineering makes it possible to precisely replicate the human genetic lesions into the Drosophila homologous genes. Finally, by systematically generating founder knock-out lines for conserved and essential genes in the long term, we will transform Drosophila into a far more efficient and versatile genetic model organism that will also be vastly more accessible and attractive to non-fly researchers for addressing their biological questions. PUBLIC HEALTH RELEVANCE: Drosophila is a leading genetic model system for addressing crucial biological questions in human diseases. Genomic engineering is a new powerful genetic tool that will significantly facilitate the analysis of complex disease pathways and the construction of better disease models in Drosophila.