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

描述(申请人提供)：RNA干扰作为哺乳动物细胞中转录后基因功能沉默的一种机制的出现，代表着基因功能实验分析的巨大进步和巨大机遇(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便捷的遗传操作方面取得了显着的进展。随着多物种siRNAs和shRNAs全基因组文库的不断发展，我们已经进入了一个新的时代，在这个时代，强大的正向遗传方法的应用可以在体内和体外使用来分配基因的功能，描绘这些基因影响正常和疾病细胞过程的分子途径，并为开发新的和改进现有的治疗方法贡献必要的知识。杜克大学RNA生物学中心、杜克综合癌症中心和应用基因组学与技术中心是杜克大学基因组科学与政策研究所的一个组成部分，它们联合起来建立了一个RNAi共享资源设施。RNAi设施的主要任务是开发和部署RNAi技术，以支持杜克大学的功能基因组研究计划。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，为研究人员提供访问全基因组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。在这项提案中，我们寻求支持，通过收购用于高通量、高含量筛选的分子器件等细胞激光扫描平板细胞仪(HT-HCS)来显著增强这一技术基础设施。IsoCyte的HT-HCS功能将基于细胞的高含量分析的能力与真正的高通量工作流结合在一起。IsoCyte的HCS技术在使用传统的基于细胞的检测时实现了超高通量的工作流程，同时提供了检测线虫和斑马鱼等大型多细胞生物的能力。此外，该仪器独特的结构和扫描速度允许使用各向异性的Fvrster共振能量转移(FRET)在单个细胞中实时执行相互作用研究。当与上游RNAi分析自动化和下游信息学相结合时，HT-HCS代表着巨大的技术进步，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.