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

被引：281

作者：

Neumann, B

Held, M

Liebel, U

Erfle, H

Rogers, P

Pepperkok, R

Ellenberg, J ^{[1
]}

机构：

[1] European Mol Biol Lab, MitoCheck Project Grp, D-69117 Heidelberg, Germany

[2] European Mol Biol Lab, Cell Biol Biophys Programme, D-69117 Heidelberg, Germany

[3] European Mol Biol Lab, Gene Express Programmes, D-69117 Heidelberg, Germany

来源：

NATURE METHODS | 2006年 / 3卷 / 05期

关键词：

D O I：

10.1038/NMETH876

中图分类号：

Q5 [生物化学];

学科分类号：

071010 ; 081704 ;

摘要：

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.

引用

页码：385 / 390

页数：6

共 22 条

[1] A large-scale RNAi screen in human cells identifies new components of the p53 pathway [J].