Figure 2
GRIM.miRNAoff vectors are turned 'on' by Cre recombinase. (A) GFP epifluorescence in HEK293 cells demonstrates GRIM.miGFPon functionality. Cells were transfected 24 hours prior with CMV.eGFP and indicated miRNA expression plasmids. GFP knockdown was evident in cells transfected with the traditionally cloned U6.miGFP plasmid or its GRIM.miGFPon counterpart, compared to cells containing an empty U6 promoter (U6 control). In contrast, GRIM.miGFPoff did not cause GFP gene silencing. Images shown are representative of three independent experiments. (B) βgal assay confirms the Cre-inducibility of the GRIM system. HEK293 cells were transfected with CMV.LacZ and indicated miRNA or control expression plasmids. Only the traditionally cloned U6.miLacZ and the GRIM.miLacZon analog caused statistically significant LacZ gene silencing (ANOVA, p < 0.05). All other constructs had no impact on βgal activity. Error bars indicate standard error of the mean (s.e.m.). U6T6 is an empty U6 promoter plasmid. UNT, untransfected HEK293 cells. (C) Small transcript northern blot shows that Cre induces full-length and processed GRIM.miLacZ. In contrast, GRIM.miLacZoff vectors do not express full-length or processed miRNAs. +, indicates a positive control DNA oligo containing the miLacZ antisense sequences. -, indicates negative DNA control corresponding to the miLacZ sense strand. A radiolabeled version of this oligo was also used to probe the blot.