Université Paris-Saclay, UVSQ, Inserm, END-ICAP, Versailles, France.
Department of Neuropediatrics, Charité-Universitätsmedizin Berlin, corporate member of Freie Universität Berlin, Humboldt-Universität zu Berlin, Berlin Institute of Health (BIH), NeuroCure Clinical Research Center, Berlin, Germany.
Neuropathol Appl Neurobiol. 2020 Oct;46(6):602-614. doi: 10.1111/nan.12639. Epub 2020 Jul 27.
Dmd , 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.
We generated a Dmd 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.
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.
Intravital imaging of Dmd muscle permits novel experimental approaches such as the study of revertant and therapeutically restored dystrophin in vivo and ex vivo.
Dmd 携带有 Dmd 外显子 23 中的 c.2983C>T 无义突变，是杜氏肌营养不良症（DMD）的小鼠模型，常用于测试旨在恢复肌营养不良蛋白的治疗方法。目前的转化研究受到缺乏报告小鼠模型的阻碍，该模型可以直接可视化肌营养不良蛋白的表达以及进行纵向体内研究。
我们构建了一个携带 mdx-23 突变和 C 端 EGFP 标签的 Dmd 报告基因等位基因。该小鼠模型允许直接可视化自发和治疗性恢复的肌营养不良蛋白-EGFP 融合蛋白，无论是在天然纤维反转后，还是例如使用三环 DNA 进行剪接调节跳过 Dmd 外显子 23 后，还是使用 AAV 编码的 CRISPR/Cas9 进行 Dmd 外显子 23 切除后。
麻醉小鼠的活体显微镜检查允许对反转纤维的肌膜肌营养不良蛋白-EGFP 融合蛋白进行实时成像，以及治疗后恢复的情况。在体外，肌营养不良蛋白-EGFP 荧光持续存在，允许对体外分离纤维中的反转和治疗性恢复的肌营养不良蛋白进行实时成像。较短的肌营养不良蛋白-EGFP 同工型 Dp71 在大脑、Dp260 在视网膜和 Dp116 在周围神经中的表达不受 mdx-23 突变的影响。
Dmd 肌肉的活体成像允许新的实验方法，例如研究体内和体外的反转和治疗性恢复的肌营养不良蛋白。
