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Figure 2 | BMC Molecular Biology

Figure 2

From: MAR-mediated integration of plasmid vectors for in vivo gene transfer and regulation

Figure 2

Regulated utrophin expression in in vivo electroporated mouse muscles. Mouse muscles were electroporated with the optimized version of Network system, consisting of the pCAG-htetR-TR450W repressor, 5Xgal4-7XtetO-Gal4VV activator and 5Xgal4-7XtetO-mUtrophin reporter constructs. The therapeutic efficacy of regulated utrophin expression in the muscles of mdx mice was assessed by atomic force microscopy [AFM, as described by Puttini et al. (2009)]. AFM assays of muscles from wild type (WT) or dystrophic (mdx) mice were performed as controls (A, B). Alternatively, 8 weeks mdx mouse muscles were assayed two weeks after the electrotransfer of the regulatory network containing the utrophin cDNA as a therapeutic transgene, and with the addition or not of doxycycline in the mice drinking water, as indicated (C, D). Muscle explant sections were immobilized and thereafter assayed using the AFM pressing mode to determine the force required to reach a given indentation in individual muscle fibers, as an assay of the expression of sufficient levels of functional utrophin to restore a normal resistance to the muscle fibers sarcolemmal membrane [29]. Results of several hundreds of independent measurements performed on distinct section locations and myofibers are represented as the distribution of Young’s modulus values in histograms constructed with a bin size of 0.5 kPa, which quantitatively describe the muscle’s resistance to mechanical deformation. Here, we pooled the results from 4 independent experiments, each one consisting of 2 electroporated animals, one treated with dox and the other one left untreated. Each animal had an 1 electrotransfer in each of the two tibialis muscles, and each muscle was processed into at least 2 sections, so a minimum of 4 muscle sections were measured per condition and experiment.

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