High-throughput RNAi screening by time-lapse imaging of live human cells
High-throughput RNAi screening by time-lapse imaging of live human cells
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DOI：
10.1038/nmeth876
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发表时间：
2006-05-01
期刊：
影响因子：
48
通讯作者：
Ellenberg, J
中科院分区：
文献类型：
--
作者：
Neumann, B;Held, M;Ellenberg, J
RNA干扰（RNAi）是研究细胞基因功能的有力工具。原则上，转染细胞微阵列允许通过显微镜进行基因敲除后的高通量表型分析。但是成像和数据分析的瓶颈限制了这种高内容筛选在固定细胞中的终点测定和全局参数（如活力）的测定。在这里，我们已经克服了这些限制，并开发了一个自动化平台，用于通过延时荧光显微镜对表达组蛋白-GFP的活HeLa细胞进行高含量RNAi筛选，以报告染色体分离和结构。我们自动化了所有步骤，包括打印准备好转染的小。干扰RNA（siRNA）微阵列、荧光成像和数字图像的计算表型分析。我们验证了这种方法在一个试点屏幕测定细胞分裂，并提供了一个敏感的，时间分辨的phenoprint的49个内源性基因，我们抑制。这个模块化平台是可扩展的，使延时显微镜的力量可用于全基因组RNAi屏幕。
RNA interference (RNAi) is a powerful toot 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.