Figure 3
GRIM.miRNAon vectors are turned 'off' by Flp recombinase. (A) GRIM.miRNAon vectors contain FRT sites flanking the U6.miRNA sequences. The entire U6.miRNA expression cassette can therefore be removed with Flp recombinase, thereby permanently switching the vector 'off'. (B and C) Testing the Flp off-switch. We generated a line of HEK293 cells stably expressing a CMV.βgal the GRIM.miLacZoff plasmids (described in Methods). Transient transfection of CMV.Cre significantly reduced βgal activity in these cells, and this silencing was maintained for up to 4 weeks (our longest timepoint) compared to the parent, unFLOXed cells (ANOVA, p < 0.05). βgal activity was significantly increased 5 days after transiently transfecting GRIM.miLacZon stable cells with the pOG44 CMV.Flp expression plasmid (ANOVA, p < 0.05), which encodes a Flp protein isoform with suboptimal activity at 37°C. Error bars indicate s.e.m.