3.1 Establishment of founder CHO cells expressing GEV transactivator
The GEV, designed as an artificial transcription factor, is a fusion protein consisting of the DNA-binding domain Gal4, ligand-binding domain ERT2 of the estrogen receptor, and transcription activation domain VP16 derived from herpes simplex virus. In the absence of E2 or 4-OHT, GEV remains in the cytoplasm; however, upon the addition of E2 or 4-OHT, GEV translocates to the nucleus through ligand binding to the ERT2 domain. Subsequently, GEV binds to the Gal4-binding sequence (UAS) of an artificial promoter (UAS-Pmin) and induces expression of the target gene under control of the artificial promoter using the activity of VP16 (Figure 1B). Initially, to establish CHO cells constitutively expressing GEV, the GEV gene was incorporated into a PiggyBac transposon vector and introduced into CHO cells along with a transposase expression vector. Stable expression cell lines were obtained by selection with the corresponding drug. As a preliminary investigation, a GFP reporter vector plasmid (PB/UAS/GFP) was transiently transfected into stable GEV-expressing cell lines, and drug-dependent GFP expression was confirmed for cells cultured in the presence of 20 µM E2 or 1.0 µM 4-OHT. Subsequently, PB/UAS/GFP was transiently introduced into these cells and cloning was performed by sorting the cell fraction induced for GFP expression under 1.0 µM 4-OHT using a cell sorter. For the obtained 26 cell clones, transient gene introduction of PB/UAS/GFP was performed, followed by culture in the presence of 1.0 µM 4-OHT. After 48 h, cell clones with high GFP expression were selected by observation under a fluorescence microscope (Supplementary Figure S1A). The same gene introduction of PB/UAS/GFP was performed for three selected cell clones. After introduction, cells were evaluated under conditions of 20 µM E2, 1.0 µM 4-OHT, and co-addition of 20 µM E2 and 1.0 µM 4-OHT (Supplementary Figure S1B). The mean fluorescence intensity of GFP was measured using a flow cytometer. In all conditions, the cell clone CHO/GEV_1 had the highest mean fluorescence intensity. Therefore, this cell clone was chosen for subsequent experiments.