Fig. 2: Splicing and potency of mirtrons located in the 5′-UTR of the eGFP transgene.
a Experimental scheme for comparison of nested and 5′-UTR mirtrons in HEK293 cells. b Rhodopsin knockdown compared for 5′-UTR mirtrons and nested equivalents. Data shown for M5 version directed against its corresponding rhodopsin species (n = 6). ns p = 0.118, *p = 0.046, ****p < 0.0001. c PCR products generated using mirtron-spanning primers and template cDNA derived from cells transfected with nested or 5′-UTR versions of mirtrons (n = 3). d Band densitometry revealed a greater proportion of correctly spliced transcripts for mirtrons located in the 5′-UTR (n = 3). Mirtron 3: p = 0.0048; Mirtron 5: p = 0.0066. e Sanger sequencing of the smallest bands (highlighted in white boxes) confirms accurate splicing of mirtrons from the 5′-UTR. Alignment of amplicon electropherograms with their corresponding reference sequences confirms absence of the 76 bp mirtron. f PCR splice analysis of tandem mirtrons (n = 3). Schematics illustrate predicted band sizes of splice products. *Band may represent a mixture of products. g Sequencing of the smallest bands in (f) confirms accurate splicing of tandem mirtrons. h Experimental scheme for comparison of M3-UTR and M3/3-UTR constructs. i Dual Glo® assay comparing M3-UTR and M3/3-UTR (n = 12). ns p = 0.0583, **p = 0.0031. j Experimental scheme for comparison of M3-UTR, M5-UTR and M3/5-UTR constructs. k Dual Glo® assay comparing M3-UTR (n = 12), M5-UTR (n = 6) and M3/3-UTR (n = 12). *p = 0.0192, ****p < 0.0001. Data for each version of M5-UTR and M3/5-UTR directed against its corresponding rhodopsin species are shown. M3/5M-UTR did not provide additional knockdown of human RHO beyond that of M3-UTR. Similarly, no difference in mouse Rho suppression was detected between M3/5H-UTR and M3-UTR. Mean ± SEM plotted throughout. Two-sided Šidák’s multiple comparisons tests used in all cases.
