A Comprehensive Human cDNA Library For Functional Gene Replacement in Drosophila

项目总结 这项提议的总体目标是开发一个工具包，旨在促进人类的功能注释 利用果蝇基因进行遗传学研究。在果蝇身上可以研究许多进化上保守的基因 使用一个简单的策略。首先，在目的基因中插入一个T2a-GAL4-PolyA人工外显子。这可以是 通过用T2a-GAL4-Polya或直接转换模拟转座元件的插入位点来完成 使用CRISPR集成此盒式磁带。这些插入通常会造成功能突变的严重损失。 此外，GAL4反式激活因子在同一组织中同时表达 利息。然后，这可以用来测试苍蝇或同源人类cDNA是否能够拯救 与苍蝇基因丢失相关的表型。如果人类的cDNA得救，人们已经确定 两个基因是同源的。然后可以确定感兴趣的人类变体(点突变)的效果 和多态)，这种方法已经被证明是非常 对人类疾病的研究很有价值。为了系统地执行这些实验，我们需要产生一个 可以在苍蝇中表达的人类cDNA文库。我们计划创建一个资源来表达~8,000 表位标记了人类和果蝇之间保守的基因的cDNA。这些cDNA 在UAS-Gal4系统的控制下，利用ϕC31整合酶将其插入到特定的基因座上。我们 将通过果蝇基因组资源中心向研究人员提供这个文库。此外，我们 将使用以下标准从这些(1500)中的子集生产转基因果蝇：与 已知的人类疾病，带有苍蝇同源物的基因，可以很容易地用现有工具操纵，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 允许果蝇和人类研究人员测试某些基因的功能替代 信息已经可用。相应的种群将存放在布鲁明顿果蝇 股票中心。最后，我们将使用T2A-GAL4-POLYA策略来为 大约有500个果蝇基因可以进行模拟插入。我们将评估一个表型 并测试人类的cdna是否能够挽救大约20例的观察到的表型。这 将确定该战略的效果如何，并为社区提供有价值的数据。我们的目标是提供 为苍蝇和人类遗传学家提供分子、遗传和转基因资源以加速鉴定 人类基因的功能。

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.