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

• 批准号: 9276156
• 负责人: HUGO J BELLEN
• 金额: $ 66.89万
• 依托单位: BAYLOR COLLEGE OF MEDICINE
• 依托单位国家: 美国
• 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/9276156
• 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; Disease; Drosophila genome; Drosophila genus; Drosophila melanogaster; Epitopes; Exons; Gene Transfer Techniques; Genes; Genetic; Genetic Polymorphism; Genetic study; Genomics; Goals; Hereditary Disease; Human; Human Genome; Injectable; 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; clinical diagnostics; cost; design; exome sequencing; experimental study; 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; site-specific integration; success; tool; vector; web site; whole genome

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.

项目摘要 该提案的总体目标是开发一个工具包，旨在促进人类的功能注释， 使用果蝇遗传学研究的基因。许多进化上保守的基因可以在果蝇中进行研究 使用一个简单的策略。首先，在感兴趣的基因中插入T2 A-GAL 4-polyA人工外显子。这可以 通过用T2 A-GAL 4-polyA转换MiMIC转座因子的插入位点或通过直接 使用CRISPR整合该盒。这些插入通常会产生强烈的功能丧失突变。 此外，GAL 4反式激活因子在相同的组织中表达，并且与GAL 4基因同时表达。 兴趣然后，这可以用于测试苍蝇或同源的人cDNA是否能够拯救昆虫。 与果蝇基因缺失相关的表型。如果人的cDNA拯救，人们已经确定， 有两个基因是正向的。然后可以确定感兴趣的人类变体（点突变）的影响 和多态性）在苍蝇中的功能性，这种方法已经被证明是非常 对人类疾病的研究很有价值。为了系统地进行这些实验，我们需要制作一个 可以在苍蝇中表达的人类cDNA库。我们计划创建一个资源，用于表达约8，000 表位标记的人cDNA的基因在人和果蝇之间是保守的。这些cdna 在UAS-GAL 4系统的控制下，将被插入到一个特定的基因座中，使用的整合酶是C31。我们 将通过果蝇基因组学资源中心向研究人员提供该文库。另外我们 将从其中的一个子集（1，500）中产生转基因果蝇，使用以下标准： 已知的人类疾病，具有苍蝇同源物的基因，可以用可用的工具轻松操纵，以及 其他研究人员（果蝇生物学家和人类遗传学家）优先考虑的基因。这些实验将 这使得果蝇和人类研究人员能够测试基因的功能替换， 目前已有资料。相应的股票将存放在布卢明顿果蝇 库存中心。最后，我们将使用T2 A-GAL 4-polyA策略来产生功能缺失突变， 大约500个果蝇基因，其中MiMIC插入是可用的。我们将评估一个人的表型， 这些的子集，并测试人cDNA是否能够拯救约20例中观察到的表型。这 将确定该战略的运作情况，并为社会提供宝贵的数据。我们的目标是提供 为苍蝇和人类遗传学家提供分子、遗传和转基因资源， 人类基因功能