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MUSCLE CELL BIOLOGY AND CELL MOTILITY

Muscle electrotransfer as a tool for studying muscle fiber-specific and nerve-dependent activity of promoters

Institut National de la Santé et de la Recherche Médicale U567, Centre National de la Recherche Scientifique Unité Mixte de Recherche 8104, Institut Cochin, Département Génétique, Développement et Pathologie Moléculaire, Université René Descartes Paris V, 75014 Paris, France

Submitted 19 March 2003 ; accepted in final form 26 June 2003

Muscle electrotransfer has recently become a promising tool for efficient delivery of plasmids and transgene expression in skeletal muscle. This technology has been mainly applied to use of muscle as a bioreactor for production of therapeutic proteins. However, it remains to be determined whether muscle electrotransfer may also be accurately used as an alternative tool to transgenesis for studying aspects of muscle-specific gene control that must be explored in fully mature muscle fibers in vivo, such as fiber specificity and nerve dependence. It was also not known to what extent the initial electrical stimulations alter muscle physiology and gene expression. Therefore, optimized conditions of skeletal muscle electroporation were first tested for their effects on muscles of transgenic mice harboring a pM310-CAT transgene in which the CAT reporter gene was under control of the fast IIB fiber-specific and nerve-dependent aldolase A pM promoter. Surprisingly, electrostimulation led to a drastic but transient shutdown of pM310-CAT transgene expression concomitant with very transient activation of MyoD and, mostly, with activation of myogenin, suggesting profound alterations in transcriptional status of the electroporated muscle. Return to a normal transcriptional state was observed 7-10 days after electroporation. Therefore, we investigated whether a reporter construct placed under control of pM could exhibit fiber-specific expression 10 days after electrotransfer in either fast tibialis anterior or slow soleus muscle. We show that not only fiber specificity, but also nerve dependence, of a pM-driven construct can be reproduced. However, after electrotransfer, pM displayed a less tight control than previously observed for the same promoter when integrated in a chromatin context.

fast or slow fibers; aldolase A; MyoD; myogenin

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