Abstract
BackgroundDystrophin 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.ResultsWe generated a DmdEGFP 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.ConclusionsThe 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.Electronic supplementary materialThe online version of this article (doi:10.1186/s13395-016-0095-5) contains supplementary material, which is available to authorized users.
Highlights
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)
Duchenne muscular dystrophy (DMD) is caused by mutations in the dystrophin gene (DMD) that prevent the expression of functional dystrophin
Dmd exon 79 was modified by changing the stop codon into a leucine and inserting a FLAG-enhanced green fluorescent protein (EGFP) sequence in-frame, followed by a neomycin cassette into the 3′UTR that was flanked by two loxP sites to enable later excision
Summary
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). We found strong natural EGFP fluorescence at all expected sites of dystrophin expression in the skeletal and smooth muscle, heart, brain, and retina. 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. Dystrophin has four main functional domains: (i) the N-terminal actin-binding domain, (ii) the central rod domain, (iii) the cysteine-rich, and (iv) the C-terminal domain. Dystrophin assembles several transmembrane and cytoplasmic proteins into the dystrophin-associated protein complex (DAPC), interactions provided by the cysteine-rich and C-terminal domains. The importance of dystrophin in DAPC assembly is highlighted by the loss of DAPC components in DMD muscle
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