( (Invitrogen). Cells were transfected wi...)siRNA screening of the cell cycle with two dynamic GFP sensors (from Discovery Matters Issue 1, July 2005)

(Invitrogen). Cells were transfected with siRNAs at 1, 5, 50, and 200 nM for 4 h followed by a media change and further incubation for 24–48 h post transfection. Cells were fixed in 4% formalin and nuclei stained with DRAQ5^™ (BioStaus).

Cellular imaging was performed on an IN Cell Analyzer 1000 using a 20× objective and 475BP20/535BP50 (GFP) and 620BP60/700BP75 (DRAQ5) excitation/emission filters. Image stacks were converted to IN Cell Analyzer 3000 format and the resulting images analyzed for cell number, cell cycle distribution, and morphology.

Results
Cellular transfection efficiency was determined using a Cy5 labeled siRNA (Fig 3). Image analysis of Cy5 distribution and intensity indicated 90% transfection efficiency. Transfection efficiency was confirmed using an siRNA pool targeting cyclin B1 sequences in the region of cyclin B1 used to construct the G2/M CCPM fusion protein (Fig 4). Treatment of cells with this siRNA pool ablated GFP fluorescence in over 90% of cells. Cells transfected with siRNAs directed against sequences present only in endogenous cyclin B1 retained EGFP expression and their fluorescence increased indicating accumulation of cells in G2 and M phase (see Fig 6, CCNB1). Measurement of cyclin B1 mRNA and EGFP fusion protein mRNA by RT-PCR and micro-array analysis (data not shown) indicated a 70% reduction in mRNA levels for both species, confirming the efficacy of the siRNA transfection.

As would be expected following knockdown of a range of cell cycle control genes, analysis of cell proliferation following siRNA treatment revealed a number of wells in which cell numbers were significantly larger or smaller than in control wells (Fig 5) indicating arrest, slowing, or acceleration of the cell cycle. For example, the effects of treating cells with siRNAs against the DNA replication licensing factor
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