To further evaluate the robustness of the CAG promoter activity, we analyzed in detail hESCs with heterozygous or homozygous targeting of a CAG-EGFP transgene in the ROSA26 or AAVS1 loci (Fig. 1A,B). For both GSHs EGFP was homogeneously expressed at high and comparable levels for more than 30 passages (Fig. S1F), and similar results were obtained after differentiation of hESCs into the three primary germ layers (Fig. S1G-M). Importantly, targeting did not interfere with pluripotency or differentiation, as shown by appropriate expression of lineage markers (Fig. S1N,O). We further differentiated these EGFP-hESC lines into fifteen different cell types (Fig. 1A), and both GSHs allowed homogeneous and strong EGFP expression in all cell types analyzed (Fig. 1C,D, Fig. S2). Overall, these results validate the ROSA26 and AAVS1 loci as suitable for robust transgene expression in both hPSCs and their derivatives.
