A Human cDNA Library for Functional Gene Replacement in Drosophila

项目总结 这项提议的目的是继续开发一个工具包，旨在促进 通过对黑腹果蝇的遗传学研究发现人类基因和疾病相关变异。我们 三年前，通过由ORIP资助的R24的支持，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。为人类建模 疾病，我们通常从创造一种苍蝇基因的严重功能丧失突变开始，这很可能是一种 已知或怀疑是致病基因的人类基因的同源基因。我们插入SA-T2A-GAL4-POLYA 利用CRISPR将人工外显子插入所有目的基因转录本(GOI)的早期内含子 介导的同源重组。这通常会产生一个强烈的功能丧失等位基因，表达 GAL4反式激活因子与突变基因具有相同的空间和时间模式。因此，无人驾驶飞机-核或 膜绿色荧光蛋白使我们能够确定基因表达的细胞类型通过与 已知的细胞身份标记或基于细胞形态。重要的是，GAL4经常允许我们拯救 与功能丧失等位基因相关的表型通过驱动UAS-Fly或人类cDNA.如果人类的cDNA 我们可以在苍蝇身上测试感兴趣的人类变体的功能，这种方法已经在很大程度上 在过去的几年里，帮助识别了许多新的人类疾病。这些实验还允许 详细的功能分析，以更好地了解致病机制并测试FDA批准的或 试验性药物。我们还制作了一个略高于2,000个T2A-GAL4库存和约3,000个无人机的库- 人类的DNA序列可以系统地进行这些实验。我们组装了一个有33,000个完整长度的图书馆 来自不同来源的人的cDNA，产生并测序了~4,000个含有UAS的质粒-人 用于在果蝇中转化的基因。其中近3000个这样的结构已经被插入到苍蝇基因组中 用ΦC31整合酶定义了基因座，并建立了转基因基因库。UAS结构包括 可从果蝇基因组资源中心(DGRC)获得，库存可从 布鲁明顿和京都股票中心。在此，我们建议扩大UAS-人基因的收集和克隆 剩余的4,000个人类cDNA中的8,500个保守基因，并建立另外3,000个转基因 用于分销的库存。我们还建议在同源Fly中产生1,000个SA-T2A-GAL4-PolA插入 使用我们开发的一种新方法来加速UAS-人类cDNA的测试 研究共同体，促进人类疾病相关基因的系统研究。我们的目标是 为苍蝇研究界和人类遗传学家提供分子、遗传和转基因资源，以 加速发现人类疾病，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.