GENOME WIDE RNAI SCREENING AND ANALYSIS

基因筛查是在模式生物中定义信号和发育途径的一种有效方法，但由于缺乏系统的工具，历史上没有被用于哺乳动物癌症生物学。最近在RNA干扰(RNAi)方面的进展已经开始促进这种方法，这些遗传工具已经成为基础和翻译癌症生物学的重要组成部分。 全基因组RNAi筛选与分析(GRSA)共享资源于2008年通过慈善和机构支持建立，以促进研究人员使用新的RNAi技术和基因筛选方法。该共享资源由Thomas Westbrook博士和Dan Liu博士指导，他们在开发基因技术(例如RNAi文库)和哺乳动物基因筛选方法方面拥有丰富的专业知识。结合这种专业知识，GRSA为全基因组遗传筛查提供了单基因分析的所有基本要素，包括全基因组范围的短发夹状rna(Shrna)。 文库、用于文库操作的多个自动化机器人平台、用于表型分析的高通量分析仪以及用于哺乳动物基因筛查的数据处理基础设施，使这一DLDCC共享资源在NCI癌症中心中独一无二。 共享资源提供的具体服务包括(1)进行全基因组或亚基因组规模的RNAi筛选，(2)利用基于慢病毒的单个shRNA载体，(3)大规模自动化操纵和制备shRNA文库，(4)自动化哺乳动物细胞转染和慢病毒生产，(5)使用自动显微镜或流式细胞仪的高通量基于细胞的分析，以及(6)数据分析、存储和管理。 通过将这些资源存放在一个单一的、有凝聚力的共享资源中，GRSA使研究人员能够使用通常成本和劳动力都高得令人望而却步的遗传方法，或者根本不适用于个别实验室。在本申请中，GRSA被建议作为DLDCC授权申请的一种新的共享资源。

Genetic screens have been a powerful approach for defining signaling and developmental pathways in model organisms, but historically have not been exploited in mammalian cancer biology because of a lack of systematic tools. Recent advances in RNA interference (RNAi) have begun to facilitate such approaches, and these genetic tools have become essential components of basic and translational cancer biology. The Genome-wide RNAi Screening and Analysis (GRSA) Shared Resource was established through philanthropic and institutional support in 2008 to facilitate investigators in their use of new RNAi technologies and genetic screening methods. The Shared Resource is directed by Drs. Thomas Westbrook and Dan Liu who have extensive expertise in developing genetic technologies (e.g. RNAi libraries) and in mammalian genetic screening methods. Combined with this expertise, the GRSA provides all essential elements for single-gene analyses to whole-genome genetic screens including genome-wide short-hairpin RNA (shRNA) libraries, multiple automated robotic platforms for library manipulation, high-throughput analyzers for phenotypic analysis, and data processing infrastructure for mammalian genetic screens, making this DLDCC Shared Resource unique among NCI Cancer Centers. Specific services provided by the Shared Resource include (1) performing whole-genome or sub-genome scale RNAi screens, (2) utilizing individual lentivirus-based shRNA vectors, (3) large-scale automated manipulation and preparation of shRNA libraries, (4) automated mammalian cell transfection and lentivirus production, (5) high-throughput cell-based assays using automated microscopy or flow cytometry, and (6) data analysis, storage, and management. By housing these resources in a single, cohesive Shared Resource, the GRSA enables investigators to employ genetic approaches that are often cost- and labor-prohibitive or simply not feasible for individual laboratories. In the current application, the GRSA is proposed as a new Shared Resource for the DLDCC grant application.