Additional file 1: Figures S1-S13. of Characterization of a Dmd
                     
                EGFP
               reporter mouse as a tool to investigate dystrophin expression
Creators: name: Petkova, Mina
name: Morales-Gonzales, Susanne
name: Relizani, Karima
name: Gill, Esther
name: Seifert, Franziska
name: Radke, Josefine
name: Stenzel, Werner
name: Garcia, Luis
name: Amthor, Helge
name: Schuelke, Markus
Publisher: name: Figshare
Year: 2016
Subjects: subject: Biochemistry
subject: Medicine
subject: Cell Biology
subject: Genetics
subject: FOS: Biological sciences
subject: FOS: Biological sciences
subject: Molecular Biology
subject: Neuroscience
subject: Physiology
subject: 39999 Chemical Sciences not elsewhere classified
subject: FOS: Chemical sciences
subject: FOS: Chemical sciences
subject: 20199 Astronomical and Space Sciences not elsewhere classified
subject: FOS: Physical sciences
subject: FOS: Physical sciences
subject: Developmental Biology
subject: 110309 Infectious Diseases
subject: FOS: Health sciences
subject: FOS: Health sciences
subject: Cardiology

description: Figure S1. Targeting of various dystrophin isoforms in Dmd EGFP mice. A schematic representation of known dystrophin isoforms 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 dystrophin at the sarcolemma. Immunofluorescent staining of cross sections from soleus (SOL), gastrocnemius (GAS), and tibialis anterior (TA) muscles of transgenic mice with anti-dystrophin antibodies 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-dystrophin antibodies. Figure S3. Correct localization of the EGFP-tagged dystrophin at the sarcolemma. Immunofluorescent staining of cross sections from soleus (SOL), gastrocnemius (GAS), and tibialis anterior (TA) muscles of transgenic mice with anti-dystrophin antibodies specific to (A) cytoskeletal β-spectrin and (B) the basement membrane protein laminin; all colored in red. Figure S4. Absence of a dystrophic phenotype in H&amp;E stained skeletal and heart muscle of adult and aged wild-type (WT) and Dmd EGFP mice. Skeletal muscles quadriceps (QUAD), gastrocnemius (GAS), extensor digitorum longus (EDL), soleus (SOL), diaphragm (DIA), and heart muscle from adult wild-type and transgenic mice show normal morphology with consistency in fiber caliber and no evidence for the presence of central nuclei, fibrosis, or the presence of inflammatory cells. No further changes are observed in aged mice in the QUAD, GAS, EDL, and heart muscle. Figure S5. Immunohistochemistry of skeletal muscles of wild-type (WT) and Dmd EGFP mice with an antibody against α-sarcoglycan. Normal sarcolemmal expression of α-sarcoglycan can be observed in the following skeletal muscles: gastrocnemius (GAS), soleus (SOL), tibialis anterior (TA), quadriceps (QUAD), and extensor digitorum longus (EDL). Figure S6. Immunohistochemistry of skeletal muscles of wild-type (WT) and Dmd EGFP mice with an antibody against β-sarcoglycan. Normal sarcolemmal expression of β-sarcoglycan can be observed in the following skeletal muscles: gastrocnemius (GAS), soleus (SOL), tibialis anterior (TA), quadriceps (QUAD), and extensor digitorum longus (EDL). Figure S7. Immunohistochemistry of skeletal muscles of wild-type (WT) and Dmd EGFP mice with an antibody against γ-sarcoglycan. Normal sarcolemmal expression of γ-sarcoglycan can be observed in the following skeletal muscles: gastrocnemius (GAS), soleus (SOL), tibialis anterior (TA), quadriceps (QUAD), and extensor digitorum longus (EDL). Figure S8. Immunohistochemistry of skeletal muscles of wild-type (WT) and Dmd EGFP mice with an antibody against α-dystroglycan. Normal sarcolemmal expression of α-dystroglycan can be observed in the following skeletal muscles: gastrocnemius (GAS), soleus (SOL), tibialis anterior (TA), quadriceps (QUAD), and extensor digitorum longus (EDL). Figure S9. Immunohistochemistry of skeletal muscles of wild-type (WT) and Dmd EGFP mice with an antibody against nNOS. Normal sarcolemmal expression of nNOS can be observed in the following skeletal muscles: gastrocnemius (GAS), soleus (SOL), tibialis anterior (TA), quadriceps (QUAD), and extensor digitorum longus (EDL). Figure S10. EGFP expression in the smooth muscle and in the hippocampus. (A) Sections from the stomach, duodenum, and colon of Dmd EGFP mice were stained with DAPI (blue). Strong natural EGFP expression (green) was detectable in the circular and longitudinal muscular layers. (B) Immunofluorescent staining of brain sections with an anti-glial fibrillary acidic protein antibody (GFAP, red) shows colocalization with the EGFP signal (green) at the glial endfeet in the hippocampus; counterstaining with 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 DAPI staining 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 counterstained with DAPI (blue). Figure S12. Western blot analysis of full-length dystrophin expression. 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 dystrophin antibody against the rod domain (MANDYS19) and against vinculin as loading control. Both wild-type and transgenic mice show comparable dystrophin protein 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 settings were used (A, B) in order to identify different dystrophin isoforms 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 dystrophin in 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. Vinculin served as loading control. WT, wild-type. (PDF 1438 kb)