A Comprehensive Resource for Manipulating the Drosophila Genome

项目总结 果蝇基因破坏计划(GDP)自2000年成立以来，已经产生了大量的、 公开可用的单个序列映射转座元件(TE)插入文库，具有 成为苍蝇研究必不可少的资源。生成和排序180,000个字节所允许的最大 有用的~22,000个(位于13,000个基因中/接近13,000个基因)将被选择并保存在布鲁明顿果蝇身上 股票中心。超过75万种GDP培养物已分发给全国数千个实验室 在国际上，促进了对数千个基因的分析。工商业污水附加费的特点 GDP，特别是模拟TE，极大地提高了它们的价值，因为它们允许对基因进行表征 表达，蛋白质分布，组织特异性击倒，相互作用蛋白的分离，评估 其他物种的同源物和其他复杂的、最先进的操作的功能。这个 灵活地将任何DNA盒交换到现有的模拟TE位点提供了无与伦比的遗传工具包， 极大地推进了功能基因组学领域，并影响了我们对基因功能的理解 物种。 在拟议的预算期间，国内生产总值将提供分析基因功能的工具， 构成了一种新的资源，不仅可以解决基本的生物学问题，还可以帮助解决 发现和研究新的人类疾病及其潜在机制。一个关键的先决条件是 在果蝇中模拟疾病的能力是表达9000个进化保守的人类中的每一个的能力 它们的果蝇同源基因的内源表达模式。目前，这可以通过使用 MIMIC和SA-T2A-GAL4-Polya盒(T2A-GAL4)。当插入两个编码之间的内含子时 外显子，这个盒是高度诱变的，并产生一种GAL4，可用于驱动一个人的UAS-cDNA 苍蝇或人类的同源物，经常挽救突变的表型，并允许疾病建模。在这里，我们 建议扩大可以用这种方法标记的大多数基因的标记。我们还有 开发了一种新的策略，允许替换所有不具有T2A-GAL4合适内含子的基因 整合，约占所有苍蝇基因的45%。这种方法交换了基因的全部编码 带有Kozak共识序列和GAL4的区域。我们建议将目前未标记的2300人作为目标 果蝇基因使用这两种策略取决于基因座的结构和基因的性质 要插入的磁带。绝大多数基因将被标记为GAL4，因为它允许许多 优雅的应用。由此产生的品系将在基因和分子上进行表征，并表达 这些基因的模式将在三龄幼虫的大脑中记录下来。这些病毒的产生和分布 试剂受到果蝇社区的高度赞赏，这从来自 苍蝇社区的领袖们。

PROJECT SUMMARY The Drosophila Gene Disruption Project (GDP), since its foundation in 2000, has produced a large, publicly available library of individual, sequence-mapped transposable element (TE) insertions that have become an essential resource for fly research. Generating and sequencing 180,000 TEs allowed the most useful ~22,000 (located in/near 13,000 genes) to be selected and deposited in the Bloomington Drosophila Stock Center. More than 750,000 GDP cultures have been distributed to thousands of labs nationally and internationally, facilitating the analysis of thousands of genes. The features of the TEs developed by the GDP, particularly the MiMIC TE, greatly enhance their value as they allow characterization of gene expression, protein distribution, tissue specific knock down, isolation of interacting proteins, assessment of the function of homologues of other species and other sophisticated, state-of-the-art manipulations. The flexibility to swap any DNA cassette into existing MiMIC TE sites provides a genetic toolkit that is unrivaled, greatly advancing the field of functional genomics and impacting our understanding of gene function across species. During the proposed budget period, the GDP will provide tools to analyze gene function that will constitute a new resource not only to tackle basic biological questions but also medical questions aiding with the discovery and study of new human diseases and their underlying mechanisms. A critical prerequisite for modeling disease in Drosophila is the ability to express each of the 9,000 evolutionarily conserved human genes in the endogenous expression pattern of their fly ortholog. This can currently be achieved by using MiMIC and the SA-T2A-GAL4-polyA cassette (T2A-GAL4). When inserted in introns between two coding exons, this cassette is highly mutagenic and produces a GAL4 that can be used to drive the UAS-cDNA of a fly or human homolog, frequently rescuing the mutant phenotype and allowing disease modeling. Here, we propose to expand the tagging of most genes that can be tagged with this approach. We have also developed a new strategy to permit replacement of all genes that do not have suitable introns for T2A-GAL4 integration, which constitute about 45% of all fly genes. This method exchanges the gene's entire coding regions with a Kozak consensus sequence followed by GAL4. We propose to target 2,300 currently untagged Drosophila genes using these two strategies depending on the structure of the locus and the nature of the cassette to be inserted. The vast majority of the genes will be tagged with GAL4 because it permits numerous elegant applications. The resulting lines will be characterized genetically and molecularly and the expression pattern of the genes will be documented in third instar larval brains. The generation and distribution of these reagents is highly appreciated by the Drosophila community as shown by the many letters of support from leaders in the fly community.