A Human cDNA Library for Functional Gene Replacement in Drosophila

项目摘要 本提案的目的是继续开发一个工具包，以便利 人类基因和疾病相关的变异通过遗传学研究在果蝇。我们 三年前，在ORIP资助的R24的支持下启动了这个项目。果蝇基因组 包含约8，500个在包括人类在内的脊椎动物中进化保守的基因。来模拟人类 疾病时，我们通常会先制造一个严重的果蝇基因功能丧失突变， 已知或怀疑致病的人类基因的直系同源物。我们插入SA-T2 A-GAL 4-polyA 使用CRISPR将人工外显子插入感兴趣基因（GOI）的所有转录物共有的早期内含子 介导的同源重组。这通常会产生一个强的功能丧失等位基因， GAL 4反式激活因子在相同的空间和时间模式的突变基因。因此，无人机系统-核或 膜GFP使我们能够确定细胞类型的基因表达，通过共染色与 已知的细胞身份标记或基于细胞形态。重要的是，GAL 4通常允许我们拯救 通过驱动UAS-fly或人cDNA与功能丧失等位基因相关的表型。如果人类cDNA 拯救了我们可以测试人类变体在苍蝇中的功能，这种方法已经大大提高了 在过去的几年里，它帮助鉴定了许多新的人类疾病。这些实验也使 详细的功能分析，以更好地了解致病机制，并测试FDA批准或 实验性药物我们还建立了一个包含2，000多个T2 A-GAL 4库存和~ 3，000个UAS的库- 人cDNA系系统地进行这些实验。我们组装了一个33,000个全长的图书馆， 来自不同来源的人cDNA，生成并测序了约4，000个含有UAS-人cDNA的质粒。 用于在果蝇中转化的cDNA。近3,000个这样的构建体已被插入到果蝇基因组中， 使用ΦC31整合酶确定的基因座，并且已经建立了转基因原种。UAS结构是 可从果蝇基因组学资源中心（Drosophila Genomics Resource Center，DGRC）获得，并且储备可从 布卢明顿和京都的股票中心。在这里，我们建议扩大UAS-人cDNA的收集和克隆 8,500个保守基因的剩余4,000个人类cDNA，并建立另外3,000个转基因 库存分配。我们还提出在同源果蝇中产生1，000个SA-T2 A-GAL 4-polyA插入物， 使用我们开发的一种新方法来加速UAS-人cDNA的测试， 研究社区和促进人类疾病相关基因的系统研究。我们的目标是 为果蝇研究界和人类遗传学家提供分子、遗传和转基因资源， 加速人类疾病的发现，帮助解开人类基因功能。

PROJECT SUMMARY The aim of this proposal is to continue to develop a toolkit designed to facilitate the functional annotation of human genes and disease associated variants through genetic studies in Drosophila melanogaster. We initiated this project three years ago through support of an R24 funded by ORIP. The Drosophila genome contains ~8,500 genes that are evolutionarily conserved in vertebrates including human. To model human diseases, we typically start by creating a severe loss-of-function mutation of a fly gene that is likely to be an ortholog of the human gene that is known or suspected to be pathogenic. We insert a SA-T2A-GAL4-polyA artificial exon into an early intron common to all transcripts of the gene of interest (GOI) using CRISPR mediated homologous recombination. This typically creates a strong loss-of-function allele that expresses the GAL4 transactivator in the same spatial and temporal pattern as the mutated gene. Hence, a UAS-nuclear or membrane GFP permits us to determine the cell types in which the gene is expressed through co-staining with known cell identity markers or based on cellular morphology. Importantly, GAL4 often allows us to rescue the phenotypes associated with the loss-of-function allele by driving a UAS-fly or human cDNA. If the human cDNA rescues we can test human variants of interest for functionality in flies, an approach that has already greatly helped in the identification of many new human diseases in the past few years. These experiments also allow detailed functional analyses to better understand the pathogenic mechanisms and to test FDA approved or experimental drugs. We have also produced a library of just over 2,000 T2A-GAL4 stocks and ~3,000 UAS- human cDNAs lines to perform these experiments systematically. We assembled a library of 33,000 full length human cDNAs from different sources, generated and sequenced ~4,000 plasmids containing the UAS-human cDNA for transformation in the fly. Nearly 3,000 of these constructs have been inserted in the fly genome in defined loci using the ΦC31 integrase, and transgenic stocks have been established. The UAS constructs are available from the Drosophila Genomics Resource Center (DGRC) and the stocks are available from the Bloomington and Kyoto stock centers. Here we propose to expand the UAS-human cDNA collection and clone the remaining 4,000 human cDNAs of the 8,500 conserved genes and establish an additional 3,000 transgenic stocks for distribution. We also propose to generate 1,000 SA-T2A-GAL4-polyA insertions in homologous fly genes using a new method that we developed to accelerate the testing of the UAS-human cDNAs by the research community and promote the systematic study of human disease associated genes. Our goal is to provide molecular, genetic and transgenic resources to the fly research community and human geneticists to accelerate the discovery of human diseases and help unravel human gene function.