IsoCyte Laser Scanning Plate Cytometer for High-throughput, High-content Assays

描述（由申请人提供）：RNA干扰作为哺乳动物细胞中基因功能转录后沉默机制的出现代表了基因功能实验分析的巨大进步和巨大机会（Hannon和Rossi，2004; Novina和Sharp，2004）。在过去的几年里，在利用RNAi途径对细胞和动物进行简单的遗传操作方面取得了显着的进展。随着多个物种的siRNA和shRNA的全基因组文库的持续开发，我们已经进入了一个新的时代，其中强大的正向遗传方法的应用可以在体内和体外用于分配基因的功能，描绘这些基因影响正常和疾病细胞过程的分子途径，并有助于开发新的和改进现有疗法所需的知识。杜克RNA生物学中心、杜克综合癌症中心以及应用基因组学和技术中心（杜克基因组科学与政策研究所的一个组成部分）已联手建立一个RNA干扰共享资源设施。RNAi设施的主要使命是开发和部署RNAi技术，以支持杜克的功能基因组学研究计划。通过对RNAi和互补技术的大量投资，我们正在为功能基因组学组装最先进的基础设施，为研究人员提供全基因组RNAi试剂和在哺乳动物细胞中进行大规模功能丧失研究所需的基础设施。在本提案中，我们寻求支持，以通过收购Molecular Devices IsoCyte激光扫描平板细胞仪来显著增强这一技术基础设施，用于高通量，高内容筛选（HT-HCS）。Isocyte联合收割机的HT-HCS功能将高含量细胞检测的功能与真正的高通量工作流程相结合。IsoCyte的HCS技术在使用传统的基于细胞的分析时能够实现超高通量工作流程，同时提供分析大型多细胞生物（如C.线虫和斑马鱼。此外，该仪器的独特结构和扫描速度允许在单个细胞中使用Fvrster共振能量转移（FRET）通过各向异性实时进行相互作用研究。当与上游RNAi检测自动化和下游信息学相结合时，HT- HCS代表了巨大的技术进步，使研究人员能够提取和量化有用的生物信息，并识别在虚拟和细胞过程中发挥作用的基因，包括疾病。

DESCRIPTION (provided by applicant): The advent of RNA interference as a mechanism for post-transcriptional silencing of gene function in mammalian cells represents a quantum advance and tremendous opportunity for experimental analysis of gene function (Hannon and Rossi, 2004; Novina and Sharp, 2004). Remarkable progress has been made over the past several years in harnessing the RNAi pathway for facile genetic manipulation of cells and animals. With the ongoing development of genome-wide libraries of siRNAs and shRNAs for multiple species, we have entered a new era in which the application of powerful forward genetic approaches can be used both in vivo and in vitro to assign function to genes, delineate molecular pathways in which these genes affect normal and disease cellular processes, and to contribute to the knowledge necessary to develop new and improve existing therapies. The Duke Center for RNA Biology, the Duke Comprehensive Cancer Center, and the Center for Applied Genomics and Technology, a component of the Duke Institute for Genome Sciences and Policy, have come together to establish an RNAi shared-resource facility. A primary mission of the RNAi Facility is to develop and deploy RNAi technologies to support functional genomics research programs at Duke. Through large investments in RNAi and complementary technologies, we are assembling a state-of-the-art infrastructure for functional genomics, providing researchers access to genome-wide RNAi reagents and the infrastructure necessary to conduct large-scale loss-of-function studies in mammalian cells. In this proposal, we seek support to significantly enhance this technology infrastructure with the acquisition of the Molecular Devices IsoCyte Laser Scanning Plate Cytometer for high-throughput, high-content screening (HT-HCS). The HT-HCS capabilities of the Isocyte combine the power of high-content cell-based assays with true high-throughput workflow. The HCS technology of the IsoCyte enables ultra-high-throughput workflows when using traditional cell-based assays, while providing the ability to assay large multicellular organisms like C. elegans and zebrafish. In addition, the unique architecture and scanning speed of this instrument allows interaction studies to be performed in real-time, in individual cells using Fvrster Resonance Energy Transfer (FRET) by anisotropy. When coupled with upstream RNAi assay automation and downstream informatics, HT- HCS represents a tremendous technological advance, enabling researchers to extract and quantify useful biological information, and to identify genes with roles in virtually and cellular processes, including disease.