Having demonstrated the suitability of the ROSA26 and AAVS1 loci for transgene expression, we developed a TET-ON inducible knockdown system based on dual GSH targeting (Fig. 2A, Fig. S3A). To simplify knockdown evaluation and method optimization we generated hESC lines in which an EGFP transgene could be silenced in an inducible fashion (Fig. 2B). To achieve this we targeted: (1) a CAG-tetR expression cassette into the ROSA26 locus; and (2) a CAG-EGFP transgene plus an inducible EGFP shRNA cassette into the AAVS1 locus (Fig. 2A,B). Interestingly, we observed a strong and homogeneous decrease in EGFP fluorescence following tetracycline treatment for 5 days (>95%; Fig. 2C), thereby confirming efficient knockdown. However, a decrease in EGFP expression was also noticed in the absence of tetracycline (Fig. 2C), suggesting a significant leakiness in the expression of the shRNA and thus confirming previous reports (Henriksen et al., 2007).
