Additional file 1: Figures S1-S13. of Characterization of a Dmd EGFP reporter mouse as a tool to investigate dystrophin expression

Figure S1. Targeting of various dystrophinisoforms in Dmd EGFP mice. A schematic representation of known dystrophinisoforms and splice variants and their alternative C-termini; in Dmd EGFP mice, the FLAG-EGFP sequence is fused to the exon 79 coding sequence of Dmd. We expected successful targeting of the isoforms Dp427 (M, B, P), Dp260, Dp140, Dp116, Dp71, Dp71d, and Dp71c. The alternatively spliced variants of Dp71, namely Dp71f, Dp71Δ110, and Dp40 contain an alternative C-terminus that does not contain the coding sequence of exon 79 and hence cannot be tagged with EGFP. Dark blue squares show the alternative C-terminal sequence generated by skipping of exon 78. Due to alternative splicing, the C-terminus of Dp40 is entirely different and fails to express EGFP as well. M, muscle-specific promoter; B, brain-specific promoter; P, Purkinje cell promoter; N, N-terminus; C, C-terminus of the polypeptide chain. Figure S2. Correct localization of the EGFP-tagged dystrophinat the sarcolemma. Immunofluorescent staining of cross sections from soleus (SOL), gastrocnemius (GAS), and tibialis anterior (TA) muscles of transgenic mice with anti- dystrophinantibodies specific to (A) the C-terminal domain (Dys2), (B) the rod domain (MANDYS19); all colored in red. Exact colocalization was observed between the natural EGFP fluorescence (green) and the signals deriving from the two anti- dystrophinantibodies. Figure S3. Correct localization of the EGFP-tagged dystrophinat the sarcolemma. Immunofluorescent staining of cross sections from soleus (SOL), gastrocnemius (GAS), and tibialis anterior (TA) muscles of transgenic mice with anti- dystrophinantibodies specific to (A) cytoskeletal β- spectrinand (B) the basement membrane protein laminin; all colored in red. Figure S4. Absence of a dystrophic phenotype in HE counterstainingwith DAPI(blue). A higher magnification is shown in the right column. Figure S11. EGFP expression in the hippocampus and in the cerebellum. (A) Left: Overview with DAPIstaining of the mouse hippocampus with the CA1-3 regions and the dentate gyrus (DG). The dashed square is zoomed in on the right side. Neuronal cells ( asterisk) of the Dmd EGFP mouse show much lower full-length dystrophin-EGFP expression than the blood vessels ( arrowhead) as visualized by immunostaining with an anti-GFP antibody (green). Due to the high expression differences, the images are depicted with two different exposure times. (B) The same pattern can be observed in the cerebellum of Dmd EGFP mice stained with anti-GFP antibody (green). Dystrophin-EGFP expression in the Purkinje cells ( asterisks) can be detected only with higher exposure times. All sections are counterstainedwith DAPI(blue). Figure S12. Western blot analysis of full-length dystrophinexpression. A western blot of different amounts (50, 5, 0.5, 0.05 μg) of whole protein extracts from the TA muscle of wild-type and Dmd EGFP -mice was probed with a dystrophinantibody against the rod domain (MANDYS19) and against vinculinas loading control. Both wild-type and transgenic mice show comparable dystrophinprotein expression levels. Figure S13. Western blot analysis of dystrophin-EGFP expression in the brain. Western blot analysis of whole brain lysates using antibodies specific against the rod domain (Dys1) and the C-terminal domain (H4) of dystrophin, as well as an anti-GFP antibody. Two different contrast settingswere used (A, B) in order to identify different dystrophinisoforms in the brain, which are expressed at different levels. (A) Dp71 is detected using the H4 antibody in the wild-type samples. The targeted isoform Dp71-EGFP shows a band running at 100 kDa detected by the anti-GFP antibody. (B) Higher contrast enhancement of the blot show Dp427 full-length dystrophinin wild-type and transgenic samples using the Dys1 antibody. The anti-GFP antibody also detected the targeted full-length form. Dp140 is detected using the H4 antibody in the wild-type samples. The targeted isoform Dp140-EGFP shows a band running at 170 kDa which is detected by the anti-GFP antibody. Vinculinserved as loading control. WT, wild-type. (PDF 1438 kb)