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

Abstract : 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.
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https://hal.archives-ouvertes.fr/hal-02984274
Contributor : Isabelle Richard Connect in order to contact the contributor
Submitted on : Monday, November 29, 2021 - 4:34:27 PM
Last modification on : Wednesday, January 19, 2022 - 1:20:02 PM

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Mina V. Petkova, Amalia Stantzou, A. Morin, O. Petrova, Susanne Morales-Gonzalez, et al.. Live-imaging of revertant and therapeutically restored dystrophin in the DmdEGFP-mdx mouse model for Duchenne muscular dystrophy. Neuropathology and Applied Neurobiology, Wiley, 2020, 46 (6), pp.602-614. ⟨10.1111/nan.12639⟩. ⟨hal-02984274v2⟩

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