A Genome-wide RNAi Screening Platform for the Common Lab

描述（申请人提供）：人类基因组计划的完成突出了建立数千个基因功能的需要。在正在开发的帮助注释哺乳动物基因组的工具中，使用RNA干扰（RNAi）的基因沉默具有前所未有的潜力，可以发现参与疾病状态下可能改变的基本细胞过程的新基因。然而，由于进行全基因组筛选所需的试剂和资本设备成本，大多数研究小组无法筛选基因组大小的siRNA文库。拟议研究的目标是开发一个创新的RNAi筛选平台，使该技术可用于普通研究实验室。微制造技术将用于在电穿孔就绪的单一导电光学透明基底上创建具有20，736个微孔（人类基因组容量）的微孔阵列，所述基底将包含在与现有成像系统兼容的标准孔板足迹中。将siRNA分子点在这些微孔内，并将细胞接种在上面。将使用新的电穿孔方案用感兴趣的siRNA转染培养的细胞。将使用标准图像采集系统和图像分析软件进行表型分析和基因注释。由于其简单性和低成本，该平台有可能使普通研究实验室能够筛选基因组规模的哺乳动物RNAi集合。通过使没有独特资源的研究人员能够全面地询问基因组，这种用于哺乳动物细胞的第二代全基因组RNAi筛选平台将显著促进人类基因组的注释。公共卫生相关性：使用RNA干扰（RNAi）的基因沉默具有前所未有的潜力，可以发现参与基本细胞过程的新基因，这些基因可能在疾病状态下发生改变。然而，对哺乳动物基因组大小的RNAi文库的筛选对于大多数研究小组来说是遥不可及的。我们的目标是开发一个新的RNAi筛选平台，使普通研究人员没有独特的资源，以全面的方式询问哺乳动物基因组。通过在哺乳动物细胞中进行基因组规模的siRNA筛选，该平台将促进基因组的注释，并有助于发现与疾病相关的基因。

DESCRIPTION (provided by applicant): Completion of the Human Genome Project has accentuated the need to establish the function of thousands of genes. Among the tools being developed to aid annotation of the mammalian genome, gene silencing using RNA interference (RNAi) has unprecedented potential to uncover novel genes involved in fundamental cellular processes that may be altered in disease states. Screening of genome-size siRNA libraries, however, has been out of reach for most research groups because of the reagent and capital equipment costs required to carry out genome-wide screens. The goal of the proposed research is to develop an innovative RNAi screening platform to make the technology accessible to the common research laboratory. Micro-fabrication techniques will be used to create a microwell array with 20,736 microwells (human genome capacity) on an electroporation-ready single conductive optically transparent substrate that will be contained in a standard well plate footprint compatible with existing imaging systems. siRNA molecules will be spotted within these microwells and cells seeded on top. Cultured cells will be transfected with the siRNA of interest using a novel electroporation scheme. Phenotypic analysis and gene annotation will be carried out using standard image acquisition systems and image analysis software. Because of its simplicity and low-cost, this platform has the potential to make screening of genome-scale mammalian RNAi collections accessible to the ordinary research laboratory. By enabling researchers without unique resources to interrogate the genome comprehensively, this second generation genome-wide RNAi screening platform for mammalian cells will significantly facilitate annotation of the human genome. PUBLIC HEALTH RELEVANCE: Gene silencing using RNA interference (RNAi) has unprecedented potential to uncover novel genes involved in fundamental cellular processes that may be altered in disease states. Screening of mammalian genome-size RNAi libraries, however, has been out of reach for most research groups. Our goal is to develop a novel RNAi screening platform to allow ordinary researchers without unique resources to interrogate the mammalian genome in a comprehensive manner. By making genome-scale siRNA screens in mammalian cells accessible to the general community, this platform will facilitate annotation of the genome and abet discovery of genes associated with disease.