Figure 2. Development of “gain of signal” assay and its actual application for drugs screening.

A. General steps of development of “gain of signal” HAC-based assay. First, the EGFP transgene (plasmid A158) was integrated into a random chromosome site in human HT1080 cells, providing a stable expression of the EGFP protein (cells are green). Separately, the tDNA-shEGFP-mCherry plasmid (A245) was inserted into a unique gene-loading loxP site of alphoid^tetO-HAC propagated in hamster CHO cells (cells are red). Then alphoid^tetO-HAC carrying a constitutively expressed shRNA against EGFP was transferred into human HT1080 cells with the integrated EGFP transgene. After HAC transfer and shRNA expressed, the EGFP transgene is silenced and the cells become red. B. Flow cytometry histograms illustrating EGFP fluorescence before and after drug treatment. The x-axis represents the intensity of the fluorescence, the y-axis the number of cells. The results of triplicate experiments are shown.