Protocols for cryo-sectioning and subsequent immunohistological targeting dystrophin staining

STSM Photo

Sofia Stenler, Sweden

The aim of this STSM was to return to the Wells group at the Royal Veterinary College for analysis of the muscle from the injections performed in COST-ONLINE_STSM-BM1207-20188. During that STSM, we performed experiments in mdx mice with the main purpose to get a proof of principle of the miMC as a functional vector for splice correction. We injected a high and a low dose, with three animals in each group. Immediately after the injection into the TA an electric pulse was applied. Material was injected into one leg, the other leg serving as internal control. The tissue was harvested after two weeks.

During this STSM at the RVC, Dr Wells taught me their very thorough protocol for cryo-sectioning and subsequent immunohistological targeting dystrophin staining to detect muscle fibres containing the exon skipped protein. Analysis of treated muscle compared to the untreated showed that treatment with miMC for exon-skipping of dystrophin had a positive effect on amount of dystrophin expressing fibres, but the effect was quite moderate. We believe that the time till harvest, two weeks after treatment, was not sufficiently long to allow accumulation of the protein.

The Wells group sectioning protocol enables harvesting material from the same muscle for protein analysis trough western blot and RNA extraction for detection of exon skipping with RT-PCR and RT-qPCR. As the number of positive fibres was so low, we chose not to perform western blot analysis but focus on RNA extraction and cDNA synthesis. From this material, I will be able to measure skipping of dystrophin as well as expression of the splice correcting U7snRNA itself in the tissue.

Together with Professor Wells and Dr Wells, I have discussed appropriate follow-up experiments to examine a more long term effect of the miMC, as well as comparing it to a standard plasmid vector encoding the same exon skipping cassette. I have provided material for such experiments.

Apart from all the laboratory work I have also had the opportunity to learn more about the work done in the Wells lab on DMD and see more of the methods they use, which has been very interesting. In all, this STSM has enabled a very educating and rewarding visit where I not only learned new methods to analyze exon skipping of dystrophin but also further developed the collaboration between the RVC and KI.

June 2014