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.