Fig. 1: Splicing efficiency and in vitro potency of candidate mirtron designs.
a GFP and Mirtron-GFP plasmid maps. The 76 bp mirtron is nested within the eGFP CDS dividing it into 5′ and 3′ fragments. b Fluorescence micrographs of HEK293 cells taken 48 h post-transfection with GFP and M-GFP plasmids using identical acquisition settings. Scale bar = 200 µm. Experiment repeated three times. c Fluorescence levels (mean ± SEM) of protein lysates from M-GFP transfected HEK293 samples normalised to that of the GFP-transfected control group (n = 3 each). d Representative UV agarose gel image of splice PCR products. The schematic to the right indicates the predicted origin of bands detected. Experiment repeated three times. e Relative densitometry of bands corresponding to PCR products derived from a mirtron-spanning primer pair (shown in d). Note that proportions of correctly spliced product as determined by this method (black bars) broadly reflect corresponding estimates of splicing efficiency derived from the fluorescence assay (shown in c). f Determination of mirtron-mediated rhodopsin suppression for human (n = 12) and mouse (n = 6) rhodopsin targets by the Dual Glo® luciferase assay (Promega, UK). Mean ± SEM is plotted in all cases. Note that each version of M5 was only effective at targeting the rhodopsin sequence of its corresponding species. Ordinary one-way ANOVA for effect of mirtron type on relative luciferase ratio for human target F(8, 99) = 41.9, p < 0.0001; and for mouse target F(8, 45) = 77.88, p < 0.0001; ****p < 0.0001 versus the mirtronless CAG.eGFP.WPRE plasmid, Dunnett’s multiple comparisons test.
