A Comprehensive Resource for Manipulating the Drosophila Genome

操纵果蝇基因组的综合资源

基本信息

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
项目类别：
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

项目摘要

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.

项目摘要自2000年基础以来，果蝇基因破坏项目（GDP）产生了大型，公开可用的个人，序列映射的可转座元素（TE）插入的库成为飞行研究的重要资源。生成和测序180,000个TE允许最多有用的〜22,000（位于/接近13,000个基因）将被选中并存放在果蝇果蝇中股票中心。在全国范围内已分发了超过750,000个GDP文化，并分配给数千个实验室。在国际上，促进了数千个基因的分析。 TES的特征由 GDP，尤其是模仿TE，在允许基因表征时大大提高了它们的价值表达，蛋白质分布，组织特异性敲低，相互作用蛋白的分离，评估其他物种和其他先进的最先进的操作的同源物的功能。这灵活性将任何DNA盒子交换到现有的模拟TE站点中，提供了一个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。在拟议的预算期内，GDP将提供分析基因功能的工具构成了一种新资源，不仅可以解决基本的生物学问题，还构成了协助医疗问题对新的人类疾病及其潜在机制的发现和研究。关键的先决条件在果蝇中建模疾病是表达9,000个进化保守的人的能力其苍蝇直系同源物的内源性表达模式中的基因。目前可以通过使用模拟和SA-T2A-GAL4-POLYA盒（T2A-GAL4）。当在两个编码之间插入内含子时外显子，此盒子是高度诱变的，并产生一个gal4，可用于驱动A的UAS-CDNA 飞行或人类同源物，经常营救突变表型并允许疾病建模。在这里，我们建议扩大可以使用这种方法标记的大多数基因的标签。我们也有制定了一种新策略，以允许更换所有没有适合T2A-GAL4内含子的基因整合，占所有飞基因的45％。此方法交换基因的整个编码具有Kozak共识序列的区域，然后是GAL4。我们建议针对目前未标记的2300个目标果蝇基因使用这两种策略，具体取决于轨迹的结构和本质磁带要插入。绝大多数基因将用GAL4标记，因为它允许许多优雅的应用。所得线将在遗传和分子上进行表征，表达基因的模式将记录在第三龄幼虫大脑中。这些的产生和分布果蝇社区高度赞赏试剂，如飞行社区的领导者。