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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.

项目总结