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A Genome-wide RNAi Screening Platform for the Common Lab

通用实验室的全基因组 RNAi 筛选平台

基本信息

批准号：
8310233
负责人：
Enrique Saez
金额：
$ 37.22万
依托单位：
SCRIPPS RESEARCH INSTITUTE, THE
依托单位国家：
美国
项目类别：
财政年份：
2009
资助国家：
美国
起止时间：
2009-08-01 至 2013-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8310233
关键词：
Animal Model Apoptosis Automation Bacteria Biological Assay Biological Process Biology Caenorhabditis elegans Capital Caspase Cataloging Catalogs Cell Cycle Regulation Cell Line Cell Survival Cell model Cell physiology Cells Cellular biology Chemicals Chemistry Code Collection Communities Computer software Cultured Cells DNA Damage Data Data Analyses Detection Development Disease Double-Stranded RNA Drosophila genus Electroporation Endocytosis Enzymes Equipment Erinaceidae Family Foundations Funding Gene Silencing Generations Genes Genetic Screening Genome Genomics Goals HIV Human Genome Human Genome Project Hypoxia Image Image Analysis Institutes Laboratories Laboratory Research Libraries Lipids Liquid substance Logistics Lower Organism Maintenance Mammalian Cell Methods Microscope Mitochondria Muscle Natural Immunity Organism Patients Phagocytosis Phenotype Physiological Physiology Plasmids Predisposition Preparation Process Property Proteins Publishing RNA Interference RNA Processing Reader Reagent Refractory Relative (related person)Research Research Personnel Resources Robot Role Running Sampling Scheme Screening procedure Sequence Analysis Signal Pathway Signaling Pathway Gene Small Interfering RNA Spottings System Techniques Technology Transfection Viral Virus Diseases West Nile virus Work Yeasts base circadian pacemaker cost feeding gene discovery gene function genome wide association study genome-wide high throughput screening innovation interest mammalian genome miniaturize novel pathogen protein protein interaction relating to nervous system research study small hairpin RNA tool

项目摘要

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.

描述（由申请人提供）：人类基因组计划的完成强调了建立数千个基因功能的需要。在正在开发的帮助哺乳动物基因组注释的工具中，使用RNA干扰（RNAi）进行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被改变。然而，由于进行全基因组筛选所需的试剂和资本设备成本，对大多数研究小组来说，筛选基因组大小的siRNA文库是遥不可及的。拟议研究的目标是开发一种创新的RNAi筛选平台，使该技术可用于普通研究实验室。微加工技术将用于在电穿孔就绪的单导电光学透明基板上创建具有20,736个微孔（人类基因组容量）的微孔阵列，该微孔阵列将包含在与现有成像系统兼容的标准孔板足迹中。siRNA分子将在这些微孔中被发现，并在上面播种细胞。培养的细胞将使用新的电穿孔方案转染感兴趣的siRNA。表型分析和基因注释将使用标准的图像采集系统和图像分析软件进行。由于其简单和低成本，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。这个第二代哺乳动物细胞全基因组RNAi筛选平台将极大地促进人类基因组的注释，使没有独特资源的研究人员能够全面地询问基因组。