Functional Genomic Screen using RNAi in Drosophila

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初级DNA序列中提取生物相关信息。在过去的几年里，出现了一些挖掘这些信息的方法。这些方法可分为表达基因组学、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。在一些生物中，dsRNA的引入已被证明是一种有效的工具，通过一种称为RNA干扰(RNAi)的过程来抑制基因表达。 重要的是，在培养的果蝇细胞中简单地添加dsRNA就可以通过RNAi减少或消除靶基因的表达，从而减少或消除表型复制功能丧失突变。果蝇细胞的这一特性使其有可能在384孔板上开发高通量RNAi筛选，以便在特定的基于细胞的分析中高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，我们已经开发了这样一种方法，这项建议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。具体地说，我们建议设立一个“RNAi设施”，接待到访的科学家，在经验丰富的工作人员协助下进行RNAi筛选。该设施将提供生物信息支持，帮助调查人员进行筛查并解释主要数据。此外，还将建立一个中央数据库，保存所有RNAi筛选的记录。“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，为了证明这种方法的价值，我们描述了一些基于细胞的筛选，这些筛选将识别与细胞存活、胞质分裂、细胞形状变化和免疫有关的基因。

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.