A Genome-wide RNAi Screening Platform for the Common Lab

点击翻译按钮获取中文摘要

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.