Characterization of a Dmd EGFP reporter mouse as a tool to investigate dystrophin expression

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.

Live‐imaging of revertant and therapeutically restored dystrophin in the DmdEGFP‐mdx mouse model for Duchenne muscular dystrophy

“…We previously demonstrated the presence of two separate bands at the position of Dp427 full-length dystrophin in Western blots from wild-type and from Dmd EGFP muscle [13], a result that we herein reproduced. Although both bands were positive for DYS1 antibody staining (directed against the rod domain), only the upper and more slowly migrating band was also positive for H4 antibody staining (directed against the C-terminus) and EGFP.…”

Multiplex in situ hybridization within a single transcript: RNAscope reveals dystrophin mRNA dynamics

“…The consistent size and intensity of 3’ foci allows this signal to be collected with greater ease than 5’ (where overexposure of the strong nuclear foci must be balanced against underexposure of the weaker sarcoplasmic foci, and 5’ foci in mature dp427 molecules close to nuclei may be lost amidst strong nascent 5’ staining), however 3’ foci without adjacent 5’ might also represent labelling of dp71 [ 26 ]. While not reported to be expressed in muscle fibres specifically, this isoform is expressed in blood vessel endothelia [ 49 ] and proliferating myoblasts [ 50 ], and is detected at low levels within whole muscle tissue homogenates, particularly those from young dystrophic muscle undergoing acute degeneration/regeneration [ 51 ]. qPCR confirms dp71 is modestly expressed in both healthy and dystrophic quadriceps muscle (in numbers that might account for ~50–70 additional 3’ foci per 20x image: see S8 Fig ) but ISH also places this modest expression in context: dp71 is robustly expressed, but apparently restricted to a small number of specific cells, potentially only visible in dystrophic muscle where levels of dp427 are lower (see Fig 4B , lower panels).…”

Single-transcript multiplex in situ hybridisation reveals unique patterns of dystrophin isoform expression in the developing mammalian embryo

“…Dp140 is found in the adult brain (primarily the cerebellum), but levels of this isoform are markedly higher and more widespread in embryonic neural tissues 17 , and dp140 is also found in the S- and comma-shaped bodies of the developing (but not adult) kidney 18 . Dp71 is particularly enriched in developing epithelial tissues, and within the lung and embryonic eye 19 , 20 . Moreover, while expression of this short isoform is essentially absent in mature myofibres, dp71 is found within proliferating myoblasts, only replaced by dp427 during differentiation 21 .…”

“…Histological studies, in contrast, offer high-resolution spatial detail, but face different challenges: the near-complete sequence-identity means antibodies specific for short isoforms are difficult to raise (as noted, C-terminal antibodies recognise all isoforms), and moreover many tissues express very low levels of protein, close to or below the limit of detection. Transgenic C-terminal fusion of eGFP reporters 36 permits study of expression even in-vivo , but again without isoform specificity. Similarly, transgenic insertion of beta-galactosidase cassettes after the unique first exon can provide valuable high-sensitivity expression data even in a whole-mount context (as shown with dp71 by Sarig et al 19 ), but the diffusible nature of both enzyme and dye lowers effective resolution to tissue rather than cell level, and this approach necessarily generates a knockout of the isoform in question, potentially confounding specific functional roles of the isoform.…”