title: Characterization of a Dmd (EGFP) reporter mouse as a tool to investigate dystrophin expression.
abstract: BACKGROUND
  Dystrophin is a rod-shaped cytoplasmic protein that provides sarcolemmal stability as a structural link between the cytoskeleton and the extracellular matrix via the dystrophin-associated protein complex (DAPC). Mutations in the dystrophin-encoding DMD gene cause X-linked dystrophinopathies with variable phenotypes, the most severe being Duchenne muscular dystrophy (DMD) characterized by progressive muscle wasting and fibrosis. However, dystrophin deficiency does not only impair the function of skeletal and heart muscle but may also affect other organ systems such as the brain, eye, and gastrointestinal tract. The generation of a dystrophin reporter mouse would facilitate research into dystrophin muscular and extramuscular pathophysiology without the need for immunostaining.
  
  
  RESULTS
  We generated a Dmd (EGFP) reporter mouse through the in-frame insertion of the EGFP coding sequence behind the last Dmd exon 79, which is known to be expressed in all major dystrophin isoforms. We analyzed EGFP and dystrophin expression in various tissues and at the single muscle fiber level. Immunostaining of various members of the DAPC was done to confirm the correct subsarcolemmal location of dystrophin-binding partners. We found strong natural EGFP fluorescence at all expected sites of dystrophin expression in the skeletal and smooth muscle, heart, brain, and retina. EGFP fluorescence exactly colocalized with dystrophin immunostaining. In the skeletal muscle, dystrophin and other proteins of the DAPC were expressed at their correct sarcolemmal/subsarcolemmal localization. Skeletal muscle maintained normal tissue architecture, suggesting the correct function of the dystrophin-EGFP fusion protein. EGFP expression could be easily verified in isolated myofibers as well as in satellite cell-derived myotubes.
  
  
  CONCLUSIONS
  The novel dystrophin reporter mouse provides a valuable tool for direct visualization of dystrophin expression and will allow the study of dystrophin expression in vivo and in vitro in various tissues by live cell imaging.
authors.affiliation: Department of Neuropediatrics, Charité-Universitätsmedizin Berlin, Augustenburger Platz 1, 13353 Berlin, Germany ; NeuroCure Clinical Research Center, Charité-Universitätsmedizin Berlin, Berlin, Germany.
authors.forename: Mina V
authors.initials: MV
authors.lastname: Petkova
authors.affiliation: Department of Neuropediatrics, Charité-Universitätsmedizin Berlin, Augustenburger Platz 1, 13353 Berlin, Germany ; NeuroCure Clinical Research Center, Charité-Universitätsmedizin Berlin, Berlin, Germany.
authors.forename: Susanne
authors.initials: S
authors.lastname: Morales-Gonzales
authors.affiliation: Université de Versailles St-Quentin, INSERM U1179 and LIA BAHN Centre Scientifique de Monaco, Montigny-le Bretonneux, France.
authors.forename: Karima
authors.initials: K
authors.lastname: Relizani
authors.affiliation: Department of Neuropediatrics, Charité-Universitätsmedizin Berlin, Augustenburger Platz 1, 13353 Berlin, Germany ; NeuroCure Clinical Research Center, Charité-Universitätsmedizin Berlin, Berlin, Germany.
authors.forename: Esther
authors.initials: E
authors.lastname: Gill
authors.affiliation: Department of Neuropediatrics, Charité-Universitätsmedizin Berlin, Augustenburger Platz 1, 13353 Berlin, Germany ; NeuroCure Clinical Research Center, Charité-Universitätsmedizin Berlin, Berlin, Germany.
authors.forename: Franziska
authors.initials: F
authors.lastname: Seifert
authors.affiliation: Institute of Neuropathology, Charité-Universitätsmedizin Berlin, Berlin, Germany.
authors.forename: Josefine
authors.initials: J
authors.lastname: Radke
authors.affiliation: Institute of Neuropathology, Charité-Universitätsmedizin Berlin, Berlin, Germany.
authors.forename: Werner
authors.initials: W
authors.lastname: Stenzel
authors.affiliation: Université de Versailles St-Quentin, INSERM U1179 and LIA BAHN Centre Scientifique de Monaco, Montigny-le Bretonneux, France.
authors.forename: Luis
authors.initials: L
authors.lastname: Garcia
authors.affiliation: Université de Versailles St-Quentin, INSERM U1179 and LIA BAHN Centre Scientifique de Monaco, Montigny-le Bretonneux, France.
authors.forename: Helge
authors.initials: H
authors.lastname: Amthor
authors.affiliation: Department of Neuropediatrics, Charité-Universitätsmedizin Berlin, Augustenburger Platz 1, 13353 Berlin, Germany ; NeuroCure Clinical Research Center, Charité-Universitätsmedizin Berlin, Berlin, Germany.
authors.forename: Markus
authors.identifier: 0000-0003-2824-3891
authors.initials: M
authors.lastname: Schuelke
mesh_terms: D020413:3' Untranslated Regions; D000818:Animals; D002478:Cells, Cultured; D016189:Dystrophin / Q000096:biosynthesis / Q000235:genetics*; D005091:Exons; D005786:Gene Expression Regulation; D017930:Genes, Reporter; D005838:Genotype; D049452:Green Fluorescent Proteins / Q000096:biosynthesis / Q000235:genetics*; D008810:Mice, Inbred C57BL; D008822:Mice, Transgenic; D008856:Microscopy, Fluorescence; D010641:Phenotype; D052097:Quadriceps Muscle / Q000166:cytology / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D032496:Satellite Cells, Skeletal Muscle
chemical_list: D020413:3' Untranslated Regions; D016189:Dystrophin; D011993:Recombinant Fusion Proteins; C085339:apo-dystrophin 1; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins
keywords: Dmd; Duchenne muscular dystrophy; Dystrophin; EGFP; Reporter mouse