Comprehensive Resource for the Drosophila 4th chromosome

果蝇第四染色体综合资源

基本信息

批准号：
10412965
负责人：
STUART J NEWFELD
金额：
$ 69.88万
依托单位：
ARIZONA STATE UNIVERSITY-TEMPE CAMPUS
依托单位国家：
美国
项目类别：
财政年份：
2020
资助国家：
美国
起止时间：
2020-06-01 至 2024-05-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10412965
关键词：
ANK2 gene Adult Advisory Committees Age related macular degeneration Ankyrins BCL9 gene Basic Science Behavior Biological Biological Assay Biology Brain Categories Chromosomes Clone Cells Clustered Regularly Interspaced Short Palindromic Repeats Code Collection Communities Complementary DNA Defect Development Disease Drosophila genus Drosophila melanogaster Eye FOXL1 gene Family Funding GDF8 gene Gene Expression Regulation Gene Proteins Genes Genetic Recombination Genome Germ Lines Goals Health Heart Homologous Gene Human Institutes Letters Light Long QT Syndrome Longevity Methods Modeling Molecular Molecular Analysis Molecular Genetics Muscle Mutation Mutation Analysis Neuraxis Neurons Pattern Proteins RNA Reporter Genes Research Personnel Resource Development Resources SOX5 gene Scientist System Technology Tissues United States National Institutes of Health Untranslated RNA Work Yang arm cooking fly frontier gain of function genetic analysis inhibin B innovation interest loss of function loss of function mutation nervous system disorder neurotransmission smoothened signaling pathway tool translational applications web site

项目摘要

For 125 years, Drosophila melanogaster has led the way in the genetic analysis of biological questions. The 4th chromosome is the final frontier for genetic analysis in Drosophila. Small and devoid of recombination the 4th has long been ignored. Nevertheless it contains 105 genes. 74% of the protein coding genes have human homologs and 68% of these have a disease association. For example Eyeless belongs to the PAX/RAX family where somatic loss of human RAX2 leads to age-related macular degeneration. Mutations in human ANK2, homolog of Ankyrin are the primary cause of congenital Long QT syndrome. A complete understanding of health requires the examination of these genes. To advance this effort, the PI recently generated unique chromosomes for the study of marked single cell clones (MARCM) carrying mutations on the 4th. Here he proposes to collaborate with colleagues at IU and UMN to facilitate the genetic and molecular analyses of every gene on the 4th. The Specific Aim is to generate a comprehensive resource for the Drosophila 4th chromosome. The resource will contain roughly 730 stocks divided into five collections. 1. FRT with a CRISPR mutation for each of the 79 protein coding genes for loss of function studies and MARCM (two mutations per gene = 158 stocks) 2. FRT with a CRISPR mutation for each of 26 noncoding RNAs for loss of function and MARCM (26 stocks). 3. Conversion of protein coding genes and noncoding RNAs with an existing MIMIC to T2A.GAL4:GFP and insertion of a CRIMIC that has T2A.GAL4:GFP in the remainder for fluorescent tagging, as reporter genes (protein or RNA) and gain of function studies (120 stocks). 4. Gain of function stocks composed of: a) UASt and UASp/UASz for each protein coding gene and non-coding RNA and b) UASt and UASp/UASz for the two closest human cDNAs for conserved protein coding genes and noncoding RNAs (400 stocks). 5. Balancer chromosomes and auxiliary chromosomes for clonal analyses such as FRT-GAL80 for MARCM, FRT-attP for designer clones and FRT-ovoD for germ line clones with/without UAS.FLP (20 stocks). The resource will enable: loss and gain of function assays, tissue-specific and temporal gene regulation in somatic and germ line tissues, the tracking of tagged RNA and proteins and all manner of genetic analyses. To assist in prioritizing tasks, we have an Advisory Committee and will accept community input. Characterization of new stocks takes place within the molecular genetics expertise of the investigators. In addition to facilitating basic research on development and disease, our resource will have direct translational application to understanding human health. For example, T2A.GAL4:GFP insertions that disrupt the fly gene and result in the expression of GAL4 in native patterns can be combined with UAS human cDNA stocks for the analysis of “humanized” disease or treatment models. This resource will be made readily available to researchers to advance our understanding of biology and conserved molecular mechanisms underlying human health.

125 年来，果蝇在生物学问题的遗传分析领域一直处于领先地位。染色体是果蝇遗传分析的最后边界，第四条染色体较小并且没有重组。然而它却含有人类74%的蛋白质编码基因，长期以来一直被忽视。同源物，其中 68% 与疾病相关，例如 Eyeless 属于 PAX/RAX 家族。人类 RAX2 的体细胞缺失会导致人类 ANK2 的年龄相关性黄斑变性，锚蛋白同源物是先天性长 QT 综合征的主要原因。为了推进这项工作，PI 最近生成了独特的基因。用于研究携带 4 号突变的标记单细胞克隆 (MARCM) 的染色体。建议与印第安纳大学和UMN的同事合作，促进遗传和分子分析 4号的每个基因的具体目标是为果蝇4号生成综合资源。该资源将包含大约 730 个种群，分为五个集合 1. 使用 CRISPR 的 FRT。用于功能丧失研究和 MARCM 的 79 个蛋白质编码基因中每一个的突变（每个突变有两个突变）基因 = 158 个库存） 2. FRT 对 26 个非编码 RNA 中的每一个进行 CRISPR 突变，导致功能丧失和 MARCM（26 个库存） 3. 将蛋白质编码基因和非编码 RNA 与现有 MIMIC 转换为 T2A.GAL4:GFP 并在其余部分插入具有 T2A.GAL4:GFP 的 CRIMIC 以进行荧光标记，作为报告基因（蛋白质或RNA）和功能获得研究（120 个股票） 4. 功能股票获得。组成：a) 每个蛋白质编码基因和非编码 RNA 的 UASt 和 UASp/UASz，以及 b) UASt 和 UASp/UASz 用于保守蛋白编码基因和非编码 RNA 的两个最接近的人类 cDNA（400 5. 用于克隆分析的平衡染色体和辅助染色体，例如用于克隆分析的FRT-GAL80 MARCM、FRT-attP 用于设计克隆，FRT-ovoD 用于带有/不带有 UAS.FLP 的种系克隆（20 个库存）。该资源将实现：功能测定的丧失和获得、组织特异性和时间基因调控体细胞和种系组织、标记 RNA 和蛋白质的追踪以及各种遗传分析。协助确定任务优先级，我们设有咨询委员会，并将接受社区的意见。除了促进研究人员的分子遗传学专业知识之外，新股票的研究也是如此。发展和疾病的基础研究，我们的资源将直接转化应用例如，T2A.GAL4：GFP 插入会破坏果蝇基因并导致 GAL4 在天然模式中的表达可以与 UAS 人类 cDNA 储备相结合，用于分析 “人性化”疾病或治疗模型将可供研究人员随时使用。增进我们对生物学和人类健康的保守分子机制的理解。