Figure 5
Correction of type VII collagen in RTM28-transduced patient keratinocytes. (a) Immunofluorescene staining of a wild-type human keratinocyte line (1, 4), an RDEB keratinocyte line (2, 5) and an RTM28-transduced RDEB keratinocyte line (3, 6). The upper panel shows type VII collagen staining using an antibody against the NC1 domain of type VII collagen, whereas staining against the FLAG tag is shown in the lower panel. Staining with the type VII collagen-specific antibody resulted in a strong signal in wild-type human keratinocytes (1), while the staining was absent in the untransduced RDEB keratinocyes (2). RTM28-transduced RDEB keratinocytes displayed a specific staining with the anti-type VII collagen antibody, corresponding to corrected type VII collagen expression upon trans-splicing (3). As expected, wild-type keratinocytes (4) and RDEB keratinocytes (5) did not show a specific signal using an anti-FLAG tag antibody, whereas staining of RTM28-transduced RDEB keratinocytes (6) resulted in a specific signal (green) in the cytoplasm of the cells. The cell nuclei were counterstained with DAPI (blue). Scale bar: 50 μm. (b) Western blot analysis of cell lysates using either an antibody against type VII collagen (upper panel) or the FLAG tag (lower panel). Wild-type human keratinocytes showed a strong type VII collagen band (1), whereas there was no band detectable in untransduced RDEB keratinocytes (2). RTM28-transduced RDEB keratinocytes (3) showed a weak band at the correct size of ~290 kDa, indicating accurate trans-splicing. Further, no band was detectable in wild-type keratinocytes (4) and untransduced RDEB keratinocytes (5) using a FLAG-specific antibody, whereas a specific ~293 kDa FLAG-tagged trans-spliced type VII collagen was visible in lysates of RTM28-transduced RDEB keratinocytes (6). Actinin staining served as loading control.
