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

Background Dmdmdx, harbouring the c.2983C>T nonsense mutation in Dmd exon 23, is a mouse model for Duchenne muscular dystrophy (DMD), frequently used to test therapies aimed at dystrophin restoration. Current translational research is methodologically hampered by the lack of a reporter mouse model, which would allow direct visualization of dystrophin expression as well as longitudinal in vivo studies. Methods We generated a DmdEGFP‐mdx reporter allele carrying in cis the mdx‐23 mutation and a C‐terminal EGFP‐tag. This mouse model allows direct visualization of spontaneously and therapeutically restored dystrophin‐EGFP fusion protein either after natural fibre reversion, or for example, after splice modulation using tricyclo‐DNA to skip Dmd exon 23, or after gene editing using AAV‐encoded CRISPR/Cas9 for Dmd exon 23 excision. Results Intravital microscopy in anaesthetized mice allowed live‐imaging of sarcolemmal dystrophin‐EGFP fusion protein of revertant fibres as well as following therapeutic restoration. Dystrophin‐EGFP‐fluorescence persisted ex vivo, allowing live‐imaging of revertant and therapeutically restored dystrophin in isolated fibres ex vivo. Expression of the shorter dystrophin‐EGFP isoforms Dp71 in the brain, Dp260 in the retina, and Dp116 in the peripheral nerve remained unabated by the mdx‐23 mutation. Conclusion Intravital imaging of DmdEGFP‐mdx muscle permits novel experimental approaches such as the study of revertant and therapeutically restored dystrophin in vivo and ex vivo.