Figure 5: Transduction and differentiation of DMD myoblasts with a lentiviral vector containing full-length dystrophin.

(a) Staining of differentiated myotubes derived from DMD myoblasts with anti-dystrophin antibody shows successful restoration of dystrophin in a GFP-sorted cell population. This staining was absent from myotubes derived from untransduced controls. Scale bar = 25 μm. (b) Staining of differentiated DMD myotubes with anti-GFP, anti-dystrophin or anti-MF20 (myosin marker) antibodies shows successful restoration of dystrophin in differentiated myotubes. Dystrophin staining is located at the sarcolemma of MF20-positive myotubes, demonstrating successful functionality of the dystrophin transgene. This staining pattern is not observed in untransduced controls. Scale bar = 25 μm. (c) The fusion index of dystrophin-transduced myoblasts closely resembles that of untransduced control myoblasts. Fusion index was calculated as the proportion of nuclei contained within MF20-positive myotubes, as a percentage of the total nuclei in the image. Data are expressed as median lines with 95% confidence intervals. N = 15 for each data set. (d) Western blotting of protein extracts shows expression of full-length dystrophin following lentiviral transduction. Staining with anti-dystrophin (right panel) shows that the GFP-sorted sample contains a dystrophin band matching that of a normal myoblast extract. This band is absent from untreated DMD myoblasts. Staining with anti-FLAG-tag confirms that the lentiviral transgene matches the size of full-length dystrophin and that dystrophin restoration is derived from the exogenous transgene.