通过对活的人类细胞进行延时成像进行高通量RNA干扰筛选。

High-throughput RNAi screening by time-lapse imaging of live human cells.

作者信息

Neumann Beate, Held Michael, Liebel Urban, Erfle Holger, Rogers Phill, Pepperkok Rainer, Ellenberg Jan

机构信息

MitoCheck Project Group, European Molecular Biology Laboratory, Meyerhofstrasse 1, D-69117 Heidelberg, Germany.

出版信息

Nat Methods. 2006 May;3(5):385-90. doi: 10.1038/nmeth876.

DOI:10.1038/nmeth876

PMID:16628209

Abstract

RNA interference (RNAi) is a powerful tool to study gene function in cultured cells. Transfected cell microarrays in principle allow high-throughput phenotypic analysis after gene knockdown by microscopy. But bottlenecks in imaging and data analysis have limited such high-content screens to endpoint assays in fixed cells and determination of global parameters such as viability. Here we have overcome these limitations and developed an automated platform for high-content RNAi screening by time-lapse fluorescence microscopy of live HeLa cells expressing histone-GFP to report on chromosome segregation and structure. We automated all steps, including printing transfection-ready small interfering RNA (siRNA) microarrays, fluorescence imaging and computational phenotyping of digital images, in a high-throughput workflow. We validated this method in a pilot screen assaying cell division and delivered a sensitive, time-resolved phenoprint for each of the 49 endogenous genes we suppressed. This modular platform is scalable and makes the power of time-lapse microscopy available for genome-wide RNAi screens.

摘要

RNA干扰（RNAi）是研究培养细胞中基因功能的强大工具。原则上，转染细胞微阵列可通过显微镜在基因敲低后进行高通量表型分析。但成像和数据分析方面的瓶颈将此类高内涵筛选限制在固定细胞的终点分析以及诸如活力等全局参数的测定上。在此，我们克服了这些限制，通过对表达组蛋白-GFP以报告染色体分离和结构的活HeLa细胞进行延时荧光显微镜观察，开发了一个用于高内涵RNAi筛选的自动化平台。我们在高通量工作流程中实现了所有步骤的自动化，包括打印可用于转染的小干扰RNA（siRNA）微阵列、荧光成像以及数字图像的计算表型分析。我们在一个检测细胞分裂的先导筛选中验证了该方法，并为我们抑制的49个内源基因中的每一个提供了灵敏的、时间分辨的表型印记。这个模块化平台具有可扩展性，使延时显微镜的强大功能可用于全基因组RNAi筛选。