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siRNA screening of the cell cycle with two dynamic GFP sensors (from Discovery Matters Issue 1, July 2005)

M. Kenrick, S. Hancock, S. Stubbs, and N. Thomas
GE Healthcare, The Maynard Centre, Cardiff, UK


Recent advances in siRNA methodologies and the development of high-throughput image analysis platforms such as the IN Cell Analyzer have revolutionized the functional analysis of genes and proteins. Here, we describe the application of two stable cell lines expressing green fluorescent protein (GFP)cell cycle sensors to screen a library of siRNAs directed against key cell cycle control genes. Imaging of GFP intensity and distribution within these two cell lines allows cell cycle position to be assigned by automated image analysis procedures and permits their use in the screening of drugs that block the cell cycle.


Introduction
Recent developments in RNA interference (RNAi) and small interfering RNA (siRNA) techniques for specifically modulating gene expression in a diverse range of cells and organisms (1, 2, 3) have revolutionized the functional analysis of genes and proteins. Advances in synthetic and virally encoded siRNA methodologies (4, 5) have now reached a stage where large scale RNAi screens can be applied to mammalian cells (6, 7, 8). In addition to the provision of large numbers of validated siRNAs, efficient mammalian siRNA functional screens will require information-rich model systems that allow abstraction of multi-parameter data at a level of throughput compatible with large-scale projects. Fortunately, advances in the capabilities of siRNA have been matched by the development of sophisticated fluorescence imagers and software capable of imaging and analyzing cellular events in live cells at high-throughput (9, 10). Such instrumentation enables study of complex systems by combining data from fluorescent cellular sensors with morphological parameters to provide a detailed description of the phenotypic effects of siRNAs in cellular scr
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