Functional Genomic Screen using RNAi in Drosophila

在果蝇中使用 RNAi 进行功能基因组筛选

• 批准号: 6596570
• 负责人: NORBERT PERRIMON
• 金额: $ 150.68万
• 依托单位: HARVARD MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-05-01 至 2007-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6596570
• 关键词: Drosophilidae; biotechnology; double stranded RNA; functional /structural genomics; gene expression; genetic library; genetic screening; genetic techniques; high throughput technology; image processing; informatics; nucleic acid chemical synthesis; nucleic acid sequence; phagocytosis; phenotype; technology /technique development; tissue /cell culture

DESCRIPTION (provided by applicant): The challenge generated by the various large scale genome projects is to derive biologically relevant information from the primary DNA sequences. In the past few years, a number of approaches to mine this information have emerged. These can be divided into Expression Genomics, Proteomics, Information Genomics, and Functional Genomics. Perhaps, the most promising Functional Genomics approach is based on the double strand RNA (dsRNA) methodology. In several organisms, introduction of a dsRNA has proven to be an effective tool to suppress gene expression through a process referred to as RNA interference (RNAi). Importantly, the simple addition of dsRNA to Drosophila cells in culture reduces or eliminates the expression of target genes by RNAi, thus phenocopying loss-of-function mutations. This property of Drosophila cells make it possible to develop high throughput RNAi screens in 384-well plates to efficiently and systematically test the function of all 14,000 Drosophila genes in specific cell-based assays. In the past few years, we have developed such a methodology and the goal of this proposal is to establish an infrastructure within the Institute of Chemistry and Cell biology at Harvard Medical School (ICCB/HMS) that will make this approach available to the entire community. Specifically, we propose to establish an "RNAi facility" where visiting scientists will be hosted to conduct RNAi screens with help of experienced staff. The facility will provide bioinformatic support to help investigators conduct the screens and interpret the primary data. In addition, a central database will be established that keeps records of all RNAi screens. The results generated by the "RNAi facility" will become a public database, and thus provide a discovery vehicle to generate hypothesis driven research. Finally, to demonstrate the value of the approach, we describe a number of cell-based screens that will identify genes involved in cell survival, cytokinesis, cell shape changes and immunity.

描述（由申请人提供）： 各种大规模基因组计划所产生的挑战是从一级DNA序列中获得生物学相关信息。在过去几年中，出现了一些挖掘这一信息的方法。这些可以分为表达基因组学，蛋白质组学，信息基因组学和功能基因组学。也许，最有前途的功能基因组学方法是基于双链RNA（dsRNA）方法。在几种生物体中，dsRNA的引入已被证明是通过称为RNA干扰（RNAi）的过程抑制基因表达的有效工具。 重要的是，在培养的果蝇细胞中简单地添加dsRNA可以通过RNAi减少或消除靶基因的表达，从而表型复制功能丧失突变。果蝇细胞的这种特性使得在384孔板中开发高通量RNAi筛选成为可能，从而在特定的基于细胞的测定中有效和系统地测试所有14，000个果蝇基因的功能。在过去的几年里，我们已经开发出这样一种方法，本提案的目标是在哈佛医学院化学和细胞生物学研究所（ICCB/HMS）内建立一个基础设施，使整个社区都可以使用这种方法。具体来说，我们建议建立一个“RNAi设施”，在那里访问科学家将在经验丰富的工作人员的帮助下进行RNAi筛选。该设施将提供生物信息学支持，以帮助研究人员进行筛选和解释原始数据。此外，还将建立一个中央数据库，记录所有RNAi筛选结果。“RNAi设施”产生的结果将成为一个公共数据库，从而提供一个发现工具来产生假设驱动的研究。最后，为了证明该方法的价值，我们描述了一些基于细胞的筛选，这些筛选将识别参与细胞存活、胞质分裂、细胞形状变化和免疫的基因。