Exon Exchange Approach to Repair Duchenne Dystrophin Transcripts
Figure 2
Exon replacement approach on dystrophin minigene transcripts.
(A) The exon exchange molecule (ExCh) AS-E24-AS' comprises the same elements as the TS molecule (see Fig. 1A) followed by a 5′ donor splice site (5′SS) and a second antisense sequence (AS') of 150 nt complementary to intron 23. The sequence of 5′SS is given in bold and is illustrated by a black ball. ExCh constructs were made with five different AS' antisense sequences, AS4 to AS8. Arrows indicate the positions of the forward A and reverse C PCR primers in the minigene. (B) Expected transcripts generated by cis-splicing (E23 inclusion and skipping) and exon exchange, and predicted sizes of the corresponding PCR amplification products detected using the RT-PCR strategy illustrated in (A). (C) RT-PCR analysis using primers A and C of NIH3T3 cells cotransfected with dystrophin minigene and constructions pSMD2 (Ctrl), pSMD2-U7-SD23-BP22 (U7), the TS constructions pSMD2-AS1-E24 (AS1), pSMD2-AS2-E24 (AS2) and ExCh molecules pSMD2-AS-E24-AS' containing AS1 or AS2 and AS4 to AS8. AS2-2XAS4, ExCh plasmid pSMD2-AS2-E24-2XAS4 containing two AS4 copies; H20: PCR negative control. Representative results from two independent transfection experiments. (D) Accurate E22-E24 and E24-E24 junctions were confirmed by sequencing of the 408bp product.