Figure 1

Exon skipping in myotube cultures derived from an exon 45 duplicated patient. A. Schematic overview of the mutation. Original and duplicated exons are shown in blue and red, respectively. Restoration of the wild type transcript can be achieved by skipping of either the original or mutated exon 45 (upper panel) but skipping of both exons 45 results in an out-of-frame transcript (lower panel). B. RT-PCR analysis. Low levels of single exon 45 skipping can be seen in the non treated sample (NT). Exon 45 skipping levels increased significantly after treatment with an exon 45 specific AON (45). Double exon 45 skipping could occasionally be observed at low levels before and after AON treatment. In-frame and out-of-frame transcripts are depicted in green and red, respectively. Duplicated exons are shaded blue. M is 100 bp size standard, C is normal control, -RT is negative control. C. Western blot analysis. Clear dystrophin (Dy4) signals were detected 2 and 5 days (2d and 5d, respectively) after AON treatment, whereas no dystrophin was observed in the non treated sample (NT). The control sample (HC) was diluted 10 times to prevent overexposure. Myosin staining (MF20) was used to confirm equal sample loading (myo). D. Immuno-histochemical analysis. No dystrophin signal was detected in non-treated myosin positive myotubes with either the Dy8 (data not shown) or the MANDYS1 antibodies (NT). After AON treatment (45AON), over 80% of myosin positive cells stained positive for dystrophin (dy8, data not shown, and MANDYS1).