Figure 2

The effect of different spacer lengths on performance of the RNAi-based OFF control device. (a) Schematic of the expression cassettes used to test the OFF control device architecture. A theophylline-responsive ribozyme switch was placed at various distances upstream of a miRNA targeting GFP. GFP was expressed constitutively under a separate expression cassette. Both constructs were stably integrated into an HEK293 Flp-In cell line. (b) GFP expression in the absence and presence of theophylline from OFF control devices with varying spacer lengths. GFP levels were measured 48 hours after the addition of 0 or 5 mM theophylline. No miR and miRNA only represent constructs with a theophylline-responsive ribozyme switch and no downstream miRNAs (negative control) and a construct with miRNA only upstream of a GFP-targeting miRNA (positive control), respectively. Data is reported as the percent of GFP expression in a cell line with no miRNA present. Error bars represent ±1 standard deviation of at least three independent experiments. (c) Relative increase in miRNA levels after addition of theophylline. Relative miRNA levels are reported 48 hours after the addition of 0 or 5 mM theophylline and normalized to the miRNA levels measured from identical cell lines in the presence of theophylline. Error bars represent ±1 standard deviation of three independent experiments.