Functional Genomic analysis using RNAi screen in Drosophila

描述（由申请人提供）：在第一轮赠款中提供的资金帮助在哈佛医学院建立了果蝇RNAi筛选中心（DRSC）。该设施的目的是为社区提供独特的基础设施和专业知识，用于使用全基因组双链RNA（dsRNA）在果蝇细胞系中进行高通量RNAi筛选（RNAi HTS）。DRSC取得了巨大的成功。在不到3年的时间里，该中心批准了来自43个不同机构的75项申请，完成了47项筛选工作，并发表了11篇详细介绍筛选工作的主要论文。在最初几年中获得的丰富经验有助于我们形成如何建立在我们最初目标的基础上并扩大果蝇中RNAi HTS的范围和技术的观点。为了面对这些新的挑战，并将DRSC发展成为功能基因组学技术转移的综合中心，我们提出以下建议：首先，通过重新设计我们的文库和提供两种或更多种dsRNA以确认RNAi表型的特异性的能力，将解决长dsRNA观察到的脱靶效应。其次，我们将实施一系列关键改进，以扩大DRSC可用的功能屏幕范围。我们将提供针对各种大基因家族（如转录因子或细胞骨架蛋白）的dsRNA的子集合以进行更集中的筛选，并将miRNA和cDNA构建体添加到过表达文库中以在果蝇细胞为基础的测定中获得功能研究。第三，为了满足对具有更大生物相关性的更复杂屏幕的日益增长的需求，我们增加了在原代果蝇细胞培养物中进行RNAi HTS的能力，并获得了超高通量共聚焦成像平台，这将是高内容成像屏幕的理想选择。第四，我们通过计划对我们的数据库进行重大升级，并开发强大的数据分析和数据挖掘计算方法，来解决筛选人员处理大量数据的需求和分析这些数据的挑战。最后，我们提供了两个实用的解决方案来验证初级RNAi筛选的结果：在哺乳动物细胞中使用siRNA进行聚焦RNAi筛选的能力，所述siRNA靶向在果蝇筛选中鉴定的基因的人类直系同源物，以及产生定制短发夹RNA转基因果蝇用于快速体内验证。

DESCRIPTION (provided by applicant): Funding provided in the first cycle of this grant helped to establish at Harvard Medical School the Drosophila RNAi Screening Center (DRSC). The purpose of the facility was to provide the community with a unique nfrastructure and expertise for high-throughput RNAi screens (RNAi HTS) in Drosophila cell lines using a genome-wide collection of double stranded RNAs (dsRNAs). The DRSC has been a resounding success. In less than 3 years, 75 applications from 43 different institutions have been approved by the center, 47 screens have been completed and 11 primary papers detailing the screens have been already published. The vast experience gained during these first years has been instrumental in shaping our view on how to build upon our initial goals and expand the scope and technology of RNAi HTS in Drosophila. To face those new challenges and develop the DRSC into an integrated center for technology transfer in functional genomics, we propose the following: First, the off-target effects observed with long dsRNAs will be addressed through a re-design of our library and the ability to offer two or more dsRNAs to confirm the specificity of RNAi phenotypes. Second, we will implement a number of key improvements to expand the range of functional screens available at the DRSC. We will offer sub-collections of dsRNAs against a variety of large gene families such as transcription factors or cytoskeletal proteins for more focused screens, and add miRNA and cDNA constructs over-expression libraries for gain of function studies in Drosophila cell- based assays. Third, to meet the growing demand on performing more sophisticated screens with greater biological relevance, we have added the ability to perform RNAi HTS in primary Drosophila cell cultures and have acquired an ultra high-throughput confocal, imaging platform that will be ideal for high content imaging screens. Fourth, we address the need of screeners to handle vast amount of data and the challenge to analyze them by planning a major upgrade of our database and developing powerful computational approaches for data analysis and data mining. Lastly, we provide two practical solutions to validate the results of the primary RNAi screen: the ability to perform focused RNAi screens in mammalian cells with siRNAs targeting human orthologs of genes identified in the Drosophila screen, and the generation of custom short hairpin RNA transgenic flies for rapid in vivo validation.