Bi-functional chimeric proteins of drug resistance genes and fluorescent markers. (A, E) Design of the vectors to test the functions of the fusion proteins driven by the CAG promoter. All constructs were electroporated into D3 mES cells, and the transfectants were selected with the standard concentrations of puromycin (A) or histidinol (D) as in Figure 1. (B, F) The numbers of the primary colonies from pac (red) and pacEGFP (green) after puromycin selection (B), and hisD (green) and hisDsRed (red) after histidinol selection. Comparable numbers of drug resistant colonies were obtained in both cases by the fusion constructs. (C) FACS analyses of the transfectants with pac vectors. EGFP fluolescence could be detected in the transfectant containing pacEGFP (green line) but not pac (red line) (upper panel: FL1). Levels of DsRed fluorescence in ES cells with pacEGFP (green line) were comparable to that with pac (red line) (lower panel: FL2). (D) Correlation between the expression levels of DsRed and Egfp from CAG-DsRed-IRES-pac-pA (upper) and CAG-DsRed-IRES-pacEGFP-pA (lower). (G) FACS analyses of the transfectants with hisD vectors. Levels of EGFP fluorescence in mES cells with hisDsRed (red line) were comparable to that with hisD (green line) (upper panel: FL1). DsRed fluolescence could be detected in the transfectant containing hisDsRed (red line) but not hisD (green line) (lower panel: FL2). (H) Correlation between the expression levels of DsRed and Egfp from CAG-EGFP-IRES-hisD-pA (upper) and CAG-EGFP-IRES-hisDsRed-pA (lower).