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. 2016 Sep 13;5(9):e362. doi: 10.1038/mtna.2016.67

Figure 5.

Figure 5

The 5′ trans-splicing strategy. (a) Schematic illustration the 5′-PTM constructs containing a CMV promoter, the wild-type Dnm2 cDNA (optimized sequence of exons 1 to 13 including exon 10 bis), a 5′ splice donor site followed by the AS and with or without polyadenylation site and intron. (b) Schematic illustration of the 5′ trans-splicing reaction between the 5′-PTM and the endogenous Dnm2 mRNA correcting the mutation in exon 11. Location of AS sequences is shown above the dotted line square. The predicted translation of the PTM (i.e., a peptide containing the Flag, the Dnm2 GTPase and Middle domain (525 aa) plus few amino acids encoded by the linker and a part of the AS sequence (59 to 64  kDa) is shown below the PTM. Primers used for RT-PCR mRNA amplification are depicted by arrows below the TS mRNA. CMV, cytomegalovirus promoter; AS, antisense sequence; SS, splices site; DISE, Downstream intronic splice enhancer; pA, polyadenylation site. PTM, pre-trans-splicing molecules; RT-PCR, reverse transcription-polymerase chain reaction; TA, Tibialis anterior.