We then sought to identify the most efficient promoter to drive constitutive transgene expression from GSHs. We tested the ability of different promoter configurations to express an enhanced green fluorescent protein (EGFP) transgene from the ROSA26 locus in hESCs (Fig. S1A,B). The highest and most homogenous EGFP expression (100%) was achieved with the artificial CAG promoter (Fig. S1C-E), which was stronger by an order of magnitude than the endogenous ROSA26 promoter (Irion et al., 2007). Interestingly, and in contrast to previous reports (Ramachandra et al., 2011), we observed that the EF1α (EEF1A1) promoter was strongly silenced, as shown by mosaic EGFP expression (Fig. S1C-E). Similar results were obtained after EGFP targeting into the AAVS1 locus (data not shown), thereby preventing the use of this promoter in subsequent experiments.
