We then hypothesized that this limitation could be bypassed by expressing higher levels of the tetR protein to more strongly repress shRNA expression in the absence of tetracycline. We performed a multi-parameter RNA and codon optimization of the bacterial tetR cDNA (Fath et al., 2011) and used the resulting codon-optimized tetR (OPTtetR) to generate new EGFP inducible knockdown hESC lines (Fig. 2B). This modification achieved a tenfold increase in tetR expression compared with the standard sequence (STDtetR; Fig. 2D). Furthermore, homozygous expression of OPTtetR was sufficient to completely prevent shRNA leakiness while fully preserving efficient knockdown induction (Fig. 2C, Fig. S3B). Of note, the inducible knockdown was rapid, reversible and dose responsive (Fig. 2E,F, Fig. S3C-E). Finally, inducible hESCs displayed a normal karyotype (data not shown), demonstrating that the genome engineering necessary to create these lines did not alter their genetic stability.
