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A Comprehensive Human cDNA Library For Functional Gene Replacement in Drosophila

用于果蝇功能基因替换的综合人类 cDNA 文库

基本信息

批准号：
9147714
负责人：
HUGO J BELLEN
金额：
$ 71.87万
依托单位：
BAYLOR COLLEGE OF MEDICINE
依托单位国家：
美国
项目类别：
财政年份：
2016
资助国家：
美国
起止时间：
2016-06-01 至 2020-05-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9147714
关键词：
Alleles Animal Model Automobile Driving Biological Clinical Clustered Regularly Interspaced Short Palindromic Repeats Collection Communities Complementary DNA DNA Transposable Elements DNA cassette Data Databases Deposition Development Diagnostic Disease Drosophila genome Drosophila genus Drosophila melanogaster Epitopes Exons Gene Transfer Techniques Genes Genetic Genetic Polymorphism Genetic study Genomics Goals Hereditary Disease Homologous Gene Human Injection of therapeutic agent Integrase Length Libraries Mediating Molecular Molecular Genetics Mus Mutagenesis Mutate Orthologous Gene Pathogenicity Pattern Phenotype Physiological Processes Point Mutation Reagent Research Research Personnel Resources Site System Technology Testing Time TimeLine Tissues Trans-Activators Transgenic Organisms United States National Institutes of Health Variant Work base cDNA Library cost design exome sequencing fly gene function gene replacement genetic variant genome sequencing human disease humanized mouse in vivo indexing insight interest loss of function loss of function mutation mutant research study site-specific integration success tool vector web site whole genome

项目摘要

PROJECT SUMMARY The overall aim of this proposal is to develop a toolkit designed to facilitate the functional annotation of human genes using Drosophila genetic studies. Numerous evolutionarily conserved genes can be studied in flies using a simple strategy. First, one inserts a T2A-GAL4-polyA artificial exon in the gene of interest. This can be done by converting the insertion sites of MiMIC transposable elements with T2A-GAL4-polyA or by direct integration of this cassette using CRISPR. These insertions typically create strong loss of function mutations. Moreover, the GAL4 transactivator is expressed in the same tissue and at the same time as the gene of interest. This can then be used to test if the fly or the homologous human cDNA is able to rescue the phenotype associated with the loss of the fly gene. If the human cDNA rescues, one has established that the two genes are orthologous. One can then determine the effect of human variants of interest (point mutations and polymorphisms) for functionality in flies, an approach that has already been shown to be extremely valuable for studies of human disease. To perform these experiments systematically we need to produce a library of human cDNAs that can be expressed in flies. We plan to create a resource for expressing ~8,000 epitope tagged human cDNAs of genes that are conserved between human and Drosophila. These cDNAs under the control of the UAS-GAL4 system will be inserted into a specific locus using the ϕC31 integrase. We will make this library available to researchers via the Drosophila Genomics Resource Center. In addition, we will produce transgenic flies from a subset of these (1,500), using the following criteria: genes associated with known human diseases, genes with fly homologs that can be easily manipulated with available tools, and genes prioritized by other researchers (Drosophila biologists and human geneticists). These experiments will allow Drosophila and human researchers to test the functional replacement of genes for which some information is already available. The corresponding stocks will be deposited in the Bloomington Drosophila Stock Center. Finally, we will use the T2A-GAL4-polyA strategy to create strong loss of function mutations for about 500 Drosophila genes for which a MiMIC insertion is available. We will assess the phenotypes of a subset of these and test if the human cDNA is able to rescue the observed phenotypes in about 20 cases. This will establish how well the strategy works and provide valuable data for the community. Our goal is to provide molecular, genetic and transgenic resources for fly and human geneticists to accelerate the identification of human gene functions.

项目总结