Exon 51 Skipping Quantification by Digital Droplet PCR in del52hDMD/mdx Mice

Methods in Molecular Biology
Monika HillerAnnemieke Aartsma-Rus

Abstract

Duchenne muscular dystrophy (DMD) is a severe, neuromuscular disorder caused by mutations in the DMD gene, precluding synthesis of functional dystrophin protein. Antisense oligonucleotide (AON)-mediated exon skipping has been developed as a method to restore the reading frame, which allows the synthesis of internally truncated, but partially functional dystrophin proteins, as found in the less severe Becker muscular dystrophy (BMD). This approach is species specific, since AONs targeting human exons often will not have full homology to mouse exons. As such, mouse models with mutations in the murine Dmd gene are of limited use to study human specific AONs in vivo. However, our del52hDMD/mdx mouse model contains mutated copies of both the mouse (nonsense mutation in exon 23) and human (deletion of exon 52) dystrophin-encoding genes. This model allows for testing effects of treatment with human specific exon 51 or 53 targeting AONs on RNA, protein, histological, and functional levels. Therefore, the model can be used to optimize human specific AONs, e.g., by comparing dystrophin protein and exon skipping levels.Absolute quantification of exon skipping levels can be obtained by digital droplet PCR (ddPCR). This method compartmental...Continue Reading

Citations

Jun 19, 2019·Annals of Clinical and Translational Neurology·Layal AntouryThurman M Wheeler

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