A Comprehensive Resource for Manipulating the Drosophila Genome

操纵果蝇基因组的综合资源

• 批准号: 10437006
• 负责人: HUGO J BELLEN
• 金额: $ 80.21万
• 依托单位: BAYLOR COLLEGE OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-07-01 至 2025-03-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10437006
• 关键词: Alleles; Basic Science; Biological; Biological Models; Brain; Budgets; C-terminal; Cells; Clustered Regularly Interspaced Short Palindromic Repeats; Code; Communities; Complementary DNA; Consensus Sequence; DNA; DNA Insertion Elements; DNA Transposable Elements; DNA cassette; Data; Deposition; Disease; Disease model; Drops; Drosophila genome; Drosophila genus; Epitopes; Exons; FlyBase; Foundations; Gene Exchanges; Gene Expression; Gene Expression Profile; Gene Proteins; Generations; Genes; Genetic; Genotype; Goals; Guide RNA; Homologous Gene; Human; Individual; Induced Mutation; Insertional Mutagenesis; Integrase; International; Introns; Length; Letters; Libraries; Maps; Mediating; Medical; Messenger RNA; Methods; Molecular; Muscle function; Mutagenesis; N-terminal; Nature; Neurons; Oogenesis; Open Reading Frames; Organism; Orthologous Gene; Pathway interactions; Pattern; Peptides; Phenotype; Polyadenylation; Proteins; RNA; RNA Splicing; Rare Diseases; Reagent; Regulation; Reporter; Research; Research Personnel; Resources; Ribosomes; Side; Signal Transduction; Site; Structure; TNFSF5 gene; Technology; Testing; Tissues; Transgenes; Variant; Viral; Yeasts; base; design; design and construction; flexibility; fly; functional genomics; gene function; gene replacement; homologous recombination; human disease; in vivo; interest; knock-down; loss of function; loss of function mutation; mutant; premature; preservation; protein distribution; synaptogenesis; tool; web site

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.

项目概要 果蝇基因破坏项目 (GDP) 自 2000 年成立以来，已产生大量、 公开的单个序列映射转座元件 (TE) 插入文库 成为苍蝇研究的重要资源。生成并测序 180,000 个 TE，允许最多 有用的约 22,000 个（位于/接近 13,000 个基因）可供选择并存放在布卢明顿果蝇中 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 在国际上，促进了数千个基因的分析。开发的TE的特点 GDP，特别是 MiMIC TE，极大地提高了它们的价值，因为它们允许表征基因 表达、蛋白质分布、组织特异性敲低、相互作用蛋白质的分离、评估 其他物种同源物的功能和其他复杂、最先进的操作。这 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的发展，并影响了我们对基因功能的理解 物种。 在拟议的预算期间，GDP 将提供分析基因功能的工具，这将 构成了一种新的资源，不仅可以解决基本的生物学问题，还可以帮助解决医学问题 新的人类疾病及其潜在机制的发现和研究。一个关键的先决条件是 果蝇疾病建模的关键是能够表达 9,000 个进化上保守的人类基因中的每一个 果蝇直系同源物的内源表达模式中的基因。目前这可以通过使用来实现 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，该盒具有高度诱变性，并产生可用于驱动 UAS-cDNA 的 GAL4 果蝇或人类同源物，经常拯救突变表型并允许疾病建模。在这里，我们 建议扩大大多数可以用这种方法标记的基因的标记。我们还有 开发了一种新策略，允许替换所有不具有适合 T2A-GAL4 内含子的基因 整合，约占所有果蝇基因的 45%。该方法交换基因的整个编码 具有 Kozak 共有序列的区域，后跟 GAL4。我们建议以 2,300 个目前未标记的目标为目标 果蝇基因使用这两种策略取决于基因座的结构和性质 要插入的磁带。绝大多数基因将被标记为 GAL4，因为它允许大量 优雅的应用程序。所得品系将在遗传和分子方面进行表征，并且表达 基因模式将记录在第三龄幼虫的大脑中。这些的产生和分配 试剂受到果蝇界的高度赞赏，从众多来自果蝇界的支持信中可以看出 苍蝇界的领导者。