Interestingly, EGFP was less reduced in cell types with slower proliferation rates (Fig. S4A). Since EGFP has an extended half-life of more than 24 h, protein loss upon transcriptional or post-transcriptional inhibition relies heavily upon its gradual dilution following cell division (Li, 1998). Considering the strong decrease in EGFP mRNA, we concluded that residual EGFP fluorescence was likely to be a consequence of the relatively short tetracycline treatment performed to trigger knockdown (5 days). To test this, we induced prolonged EGFP knockdown in postmitotic cardiomyocytes, and we indeed observed a slow but constant decrease in protein expression for up to 20 days, at which point the EGFP was decreased by more than 75% (Fig. S4H,I). To reinforce these observations, we performed similar experiments using a ROSA26-targeted EGFP reporter transgene fused to a destabilization domain (EGFPd2; Li et al., 1998), which avoids confounding effects due to the long half-life of standard EGFP (Fig. S4J-M) (Wahlers et al., 2001). Remarkably, EGFPd2 inducible knockdown in cardiomyocytes using the sOPTiKD method resulted in >90% protein knockdown after 5 days of tetracycline treatment (Fig. S4N,O). Considered together, these results establish that OPTiKD and sOPTiKD allow efficient manipulation of gene expression even after differentiation of hPSCs.
