( eens. P In this study ...)siRNA screening of the cell cycle with two dynamic GFP sensors (from Discovery Matters Issue 1, July 2005)

In this study we have used two stable cell lines expressing green fluorescent protein cell cycle sensors (Fig 1) to screen a library of siRNAs directed against key cell cycle control genes.

One cell line (11, 12) reports on G2 phase to M phase cell cycle transition via dynamic expression and degradation of an EGFP sensor that shadows endogenous cyclin B1 levels. The G2/M cell cycle phase marker (CCPM) is switched on in late S phase, switched off at the end of mitosis, and in the intervening period translocates from the cytoplasm to the nucleus.

The second cell line reports on G1 phase to S phase transition via translocation of an EGFP fusion protein incorporating the phosphorylation-dependent sub-cellular location domain (PSLD) of DNA helicase B (13). In this cell line a nuclear localization sequence (NLS) within the PSLD retains the fusion protein within the nuclei of G1 cells. Phosphorylation of serine⁹⁶⁷ within the PSLD by CDK2/cyclin E during S phase unmasks a previously inactive nuclear export sequence that predominates over the NLS leading to export of the fusion protein into the cytoplasm in S phase cells.

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 (Fig 2).

siRNA screening
Cell cycle gene knockdowns were carried out using a Dharmacon siARRAY^™ of 112 siRNA pools each comprising four siRNAs directed against a single cell cycle related gene. Additional scrambled sequence and Cy^™5 labeled siRNAs were used as controls and to determine transfection efficiency. siRNAs were transfected into G2/M CCPM and G1/S CCPM expressing U2OS cells in 96-well plates using Lipofectamine^™ 2000
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