Figure 3
From: Muscle regeneration affects Adeno Associated Virus 1 mediated transgene transcription
Impact of muscle regeneration on vector genome content and its intracellular localization. (A) Quantification of AAV genomes per nucleus by absolute qPCR in wt, wt + CTX and mdx muscles injected with AAV1-mSeAP vector. Three weeks after AAV injections, TAs were collected and analysed. Considering the higher numbers of total nuclei in wt + CTX and mdx muscles compared to wt muscles, the obtained data for these muscles were adjusted by a correcting factor (1.7 and 1.5, respectively). AAV genome content is expressed as the AAV genome number relative to the value obtained for wt muscles. Non inj., wt non-AAV injected TA. (B) Western blot analysis of total (T), nuclear (N) and cytosolic (C) fractions from the three muscle groups using GAPDH and H3 antibodies, confirming cytosolic and nuclear enrichments. EEA1 and GM130 antibodies detect markers of early endosomes and Golgi apparatus present in the cytosolic fractions validating the nuclear fraction purity. (D) AAV genomes were quantified by absolute qPCR in nuclear and total fractions from the three muscle groups and the proportion of vg present in nuclear fractions was calculated. (D) Quantification of AAV genomes per nucleus by absolute qPCR 10 month after regeneration. Considering the higher numbers of total nuclei in wt + CTX muscles compared to wt muscles, the obtained data for wt + CTX were adjusted by the correcting factor 1.7. AAV genome content is expressed as the AAV genome number relative to the value obtained for wt muscles (6w). Non inj., wt non-AAV injected TA. The data represent the mean values of five TAs per group ± SEM. n.s., non-significant, ***P ≤ 0.001.
