2.3 Generation of recombinant CHO cells
To generate CHO cells expressing the GEV gene (CHO/GEV), CHO-K1 cells were seeded in a 24-well plate at 1.0 x 105cells/well. The next day, PB/chEF1α/GEV_Hyg and PiggyBac transposase expression vector plasmids (pPBase; #PB210PA-1, System Biosciences) were transiently transfected into CHO-K1 cells using Lipofectamine 2000 (#11668019, Invitrogen) according to the manufacturer’s procedure. After 48 h, cells were plated in six-well plates (BioLite #130184, Thermo Scientific), Hygromycin B (#008-07683, Wako) was added at a concentration of 400 µg/mL, and drug selection was performed for 14 days. Stable transformed cells expressing GEV were obtained. Cell clones (CHO/GEV) were established by limiting dilution.
Next, PB/UAS/GFP_Zeo was transiently introduced into CHO/GEV together with pPBase using Lipofectamine 2000 as described above. Drug selection was performed for 14 days using 400 µg/mL Zeocin (#R25001, Invitrogen). Next, 1 µM 4-OHT (#H6278, Sigma-Aldrich) was added to the cells. The day after drug addition, single-cell cloning of GFP-positive cells was performed using a cell sorter (SH800; Sony, Tokyo, Japan) to establish drug-inducible GFP-expressing cell clones (CHO/GEV_GFP).
To generate drug-inducible scFv-Fc-producing cells, PB/UAS/scFv-Fc_Zeo linearized with Fsp I was introduced into CHO/GEV using Lipofectamine 2000. Selection with Zeocin was performed in the same manner as described above to establish stable transformed cells, and cell clones (CHO/GEV_scFv-Fc) were obtained by limiting dilution.