A Human cDNA Library for Functional Gene Replacement in Drosophila

用于果蝇功能基因替换的人类 cDNA 文库

基本信息

批准号：
10162680
负责人：
HUGO J BELLEN
金额：
$ 75.46万
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 2024-05-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10162680
关键词：
Affect Alleles Animal Model Antibodies Automobile Driving Bacterial Artificial Chromosomes CD8B1 gene CRISPR/Cas technology Cells Cellular Morphology Clinical Clustered Regularly Interspaced Short Palindromic Repeats Collection Communities Complementary DNA Data Disease Drops Drosophila genome Drosophila genus Drosophila melanogaster Epitopes Exons FDA approved Fishes FlyBase Funding Gene Expression Profile Gene Transfer Techniques Generations Genes Genetic Diseases Genetic study Genome Genomics Goals Human Human Genetics Injections Integrase Introns Length Libraries Mediating Membrane Methods Molecular Molecular Genetics Monoclonal Antibody R24 Mutagenesis Mutate Mutation Nuclear Orthologous Gene Pathogenicity Patients Pattern Pharmaceutical Preparations Phenotype Plasmids Play Proteins Publications Quick Test for Liver Function RNA Interference Reagent Research Resistance Resources Role Severities Site Source Specificity Stains Structure Technology Testing Tissues Trans-Activators Transcript Transgenic Organisms Variant Vertebrates Work Zebrafish base cDNA Library cell type clinical research site cost design experimental study fly gene discovery gene function gene replacement homologous recombination human data human disease human model in vivo in vivo evaluation insight interest knock-down loss of function loss of function mutation mutant novel online community overexpression proband protein function screening site-specific integration success vector

项目摘要

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.

项目摘要该建议的目的是继续开发一种工具包，以促进功能注释人类基因和疾病通过果蝇中的遗传研究相关的变异。我们三年前，通过支持Orip资助的R24来启动该项目。果蝇基因组包含约8,500个基因，这些基因在包括人在内的脊椎动物中在进化上保守。建模人类疾病，我们通常从创建一个可能是一个可能是一个蝇基因的严重功能丧失突变开始人类基因的直系同源物已知或怀疑是致病性的。我们插入sa-t2a-gal4-polya 使用CRISPr 介导的同源重组。这通常会产生强大的功能丧失等位基因表达与突变基因相同的空间和临时模式的GAL4反式激活器。因此，一个UAS核或膜GFP允许我们确定通过与已知的细胞身份标记或基于细胞形态。重要的是，Gal4通常允许我们营救通过驱动UAS闪光或人类cDNA与功能丧失等位基因相关的表型。如果人cDNA 营救我们可以测试苍蝇中功能性功能感兴趣的人类变体，这种方法已经很棒在过去的几年中，帮助鉴定了许多新的人类疾病。这些实验也允许详细的功能分析以更好地了解致病机制并测试已批准的FDA或实验药物。我们还生产了一个库的图书馆，其中有2,000多个T2A-GAL4股票和约3,000个UAS- 人cDNA线系统地执行这些实验。我们组装了一个全长33,000的库来自不同来源的人cDNA，生成和测序〜4,000个质粒，包含UAS-HUMAN cDNA，用于苍蝇转化。这些结构中有将近3,000个已插入了Fly基因组中使用φC31积分酶定义的基因座，已经建立了转基因库存。 UAS结构是可从果蝇基因组资源中心（DGRC）获得，可从股票中获得布卢明顿和京都股票中心。在这里，我们建议扩展UAS-Human cDNA收集和克隆 8,500个保守基因的其余4,000个人cDNA，并建立了3,000个转基因分配股票。我们还建议在同源苍蝇中生成1,000 sa-t2a-gal4-polya插入使用一种新方法，我们开发了一种新方法，以加速通过研究社区并促进对与人类疾病相关基因的系统研究。我们的目标是向FLE研究界和人类遗传学家提供分子，遗传和转基因资源加速发现人类疾病并帮助解释人类基因的功能。