Expression levels of the transgenes from the stably integrated bi-cistronic transgene cassettes with various drug selection systems. (A) Design of the bi-cistronic expression vectors containing the drug resistance genes. The drug resistance genes for G418 (neo), puromycin (pac), hygromycin B (hph), zeocin (zeo), blasticidin S (bsd), and histidinol (hisD) were fused to EMCV-IRES and placed downstream of Egfp under the control of the CAG expression unit . (B) Determination of the minimal concentrations of the drugs. The D3 mES were seeded at low cell density (1000 cells per 90 mm dish) with various concentrations of drugs in triplicate, and the cell numbers were counted. The relative number of the cells to that obtained without the drug, which was set at 1.0, are shown. Asterisks show the doses of 1× of the selection in FACS analyses, by which more than 93% of the wild-type mES cells were killed. (C) The FACS analyses for the EGFP expression levels. In these FACS analyses, the EGFP expression level (FL1) under the selection with the minimal concentration (1×, green line), twice higher than the minimal concentration (2×, red line) and three times higher than the minimal concentration (3×, blue line) were analyzed by flow cytometry using FACSCalibur (BD). Blue filled peaks indicate the signals from wild-type mES cells. (D) Quantification of the relative cell numbers expressing EGFP. The proportions within M1 indicated in C are shown.