A Comprehensive Resource for Manipulating the Drosophila Genome

项目摘要 果蝇基因破坏项目（GDP）自2000年成立以来，已经产生了大量， 公开可获得的单个序列映射的转座因子（TE）插入文库，其具有 成为苍蝇研究的重要资源。生成和测序180，000个TE， 在布卢明顿果蝇中选择和储存有用的~ 22，000个（位于/接近13，000个基因中） 股票中心。超过750，000个GDP培养物已分发到全国数千个实验室， 在全球范围内，这有助于分析成千上万的基因。开发的TE的特点 GDP，特别是MiMIC TE，大大提高了它们的价值，因为它们允许表征基因 表达，蛋白质分布，组织特异性敲除，相互作用蛋白质的分离， 其他物种的同源物的功能以及其他复杂的、最先进的操作。的 将任何DNA盒交换到现有MiMIC TE位点的灵活性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， 构成了一个新的资源，不仅要解决基本的生物学问题，而且要解决医学问题， 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， 在果蝇中模拟疾病的能力是表达9,000个进化上保守的人类基因中的每一个。 基因在其果蝇直系同源物的内源表达模式中。目前可以通过使用 MiMIC和SA-T2 A-GAL 4-polyA盒（T2 A-GAL 4）。当插入两个编码区之间的内含子时， 外显子，该盒是高度致突变的，并产生GAL 4，其可用于驱动UAS-cDNA， 苍蝇或人类同源物，经常拯救突变表型，并允许疾病建模。这里我们 建议扩大大多数可以用这种方法标记的基因的标记。我们还 开发了一种新的策略，允许替换所有没有合适的T2 A-GAL 4内含子的基因 整合，占所有果蝇基因的约45%。这种方法交换了基因的整个编码 Kozak共有序列后接GAL 4的区域。我们建议把2,300个目前没有标记的目标 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。 要插入的卡匣。绝大多数基因将被标记GAL 4，因为它允许许多 优雅的应用。将对所得品系进行遗传和分子表征， 将在三龄幼虫脑中记录基因的模式。它们的产生和分布 试剂受到果蝇社区的高度赞赏，如来自 苍蝇社区的领导者。

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.