3.2 Inducible expression of GFP reporter gene in CHO/GEV_GFP cells
We next investigated proliferation and gene expression responsiveness using a GFP reporter gene. A transposon vector containing the GEV-responsive GFP expression unit was introduced into CHO/GEV cells and integrated into the cell genome through transposition. Upon the addition of 1.0 µM 4-OHT to drug-selected cells, GFP expression was induced in all cells. Therefore, cloning was performed using a cell sorter. All six tested clones showed equivalent GFP induction and proliferation responsiveness upon 4-OHT addition. Based on these results, a representative cell clone (CHO/GEV_GFP) was used for further analysis.
CHO/GEV_GFP cells were seeded in a 24-well plate to evaluate cell proliferation and drug responses (Figure 2). Various concentrations of E2-containing medium were used, and the medium was changed daily. As a result, a concentration-dependent decrease in proliferation was observed (Figure 2A). When 20 µM E2 was added, cell proliferation was completely suppressed without decreasing cell viability. In our analysis ofGFP expression induction, GFP-positive cells were observed from the day after the addition of E2, and 80%–90% of GFP-positive cells were observed throughout the culture period at E2 concentrations of 5 µM or higher (Figure 2B). However, the mean fluorescence intensity was only about twice that of the non-additive control, suggesting that the expression level was insufficient (Figure 2C). Next, we attempted gene expression induction using 4-OHT, an antagonist of E2. Cell toxicity was observed at concentrations of 10 µM or higher (Supplementary Figure S2). Therefore, we evaluated CHO/GEV_GFP cells with a low concentration range of 0.01–1 µM 4-OHT. As a result, a decreased proliferation rate was observed and cells were able to proliferate without toxicity (Figure 2D). Analysis of GFP expression inducibility reveals that almost all cells became GFP-positive from day 1 of culture at concentrations of 0.5 µM 4-OHT or higher (Figure 2E). The mean fluorescence intensity of GFP reached its maximum on day 3 of culture, and a 2.5-fold higher expression was observed with 4-OHT compared with E2 (Figure 2F). This is attributable to the high affinity of 4-OHT for ERT2 compared with E2.[32] Thus, it was found that 4-OHT could induce higher expression at concentrations more than 10-fold lower than E2.