Figure 2.

Construct strategy of multi-fluorescent sensors and expression in vitro. Construct strategy of the three fluorescent sensors of cell cycle progress was designed as diagram. The constructs were transfected into HEK293 cells to confirm the function of expression. (a), Cyclin B1 promoter drove cre recombinase initiates the cascade reaction of the fluorescent sensors. Cyclin B1 promoter drove cyclin B1 fused EGFP with double loxP sites and Flp is a key plasmid to show proliferating cells. CMV promoter drove DsRed and EYFP fragment with double Frt sites is indicating the proliferated cell transition from red to yellow fluorescence. Cotransfected of triple plasmids would be possible to recognize the cell population with variety fluorescence. (b), CMV-DsRed–EYFP and Cyclin B1-EGFP-Flp plasmids were transfected into HEK293 cells respectively. (c), CMV-DsRed-EYFP and Cyclin B1-EGFP-Flp plasmids were cotransfected into HEK293 cells together showing G1 phase cells (red arrows) and G2 phase cells (white arrows). (d), Cyclin B1-cre, CMV-DsRed-EYFP and Cyclin B1-EGFP-Flp plasmids were cotransfected into HEK293 cells together showing the cells passed one cell cycle (red arrows) and the cells in the first cell cycle (white arrows).