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. 2011 Jun 17;39(16):e108. doi: 10.1093/nar/gkr465

Figure 4.

Figure 4.

Detection of acGFP restoration by double RNA trans-splicing on mRNA level. Trans-splicing on mRNA level can be detected by RT–PCR in HEK293AD cells co-transfected with RTM 4 or RTM 2 and target molecule expressing plasmids. COL17A1 exon 52 replacement by the internal acGFP fragment contributed by the RTM was detected using a 5′ acGFP and 3′ acGFP specific primer pair to discriminate between cis- and trans-splicing events. The cDNA templates included: lane 1: target molecule alone, lane 2: RTM 2 plasmid only, lane 3: RTM 4 plasmid only, lane 4: target molecule + RTM 2 plasmids, lane 5: target molecule + RTM 4 plasmids, lane 6: water control. The cis-splicing product 5′acGFP - exon 52 - 3′acGFP (lanes 1, 4, 5), the trans-splicing product acGFP (lanes 4 and 5; The marked band in lane 4, indicated by an arrow, was sequenced after recovery and gel extraction) or the exon skipping product 5′ acGFP - 3′ acGFP (lanes 1, 4, 5) was visualized on a 2% agarose gel after gel electrophoresis. Lane 7: DNA ladder mix (Fermentas).