Electrotransfer in differentiated myotubes: a novel, efficient procedure for functional gene transfer.
Development of reliable techniques for experimental manipulation of gene expression in multinucleated skeletal muscle fibers is critical for understanding molecular mechanisms involved in both physiology and pathophysiology. At present, viral vectors represent the only method to obtain efficient gene transfer in terminally differentiated myotubes. Here we present an in vitro procedure that relies on the application of a pulsed electric field for transferring naked DNA into differentiated myotubes seeded on coverslips. Compared with standard transfection methods, electroporation was at least 1000 times more efficient, as judged by quantitative determination of luciferase content. Percentage of transfected myotubes averaged around 45%. Moreover, we were successful in transfecting a dominant-negative ADP ribosylation factor 1 (ARF1) mutant, i.e., ARF1N126I, in myotubes, thus interfering with endoplasmic reticulum-Golgi traffic, as indicated by alterations of subcellular distribution of GM130, a cis/medial-Golgi marker. Co-transfection experiments with beta-galactosidase also showed that the ARF1 mutant appeared to inhibit myoblast fusion and could not be used before myotube formation. The present work validates the use of electroporation as a highly efficient approach for gene transfer in fully differentiated myotubes.
Dipartimento di Scienze Biomediche Sperimentali, Università di Padova, viale G. Colombo 3, 35121 Padua, Italy.
Marco
M
Sandri
Elena
E
Bortoloso
Alessandra
A
Nori
Pompeo
P
Volpe
D000818:Animals; D002454:Cell Differentiation / Q000502:physiology*; D002460:Cell Line; D048429:Cell Size; D004247:DNA / Q000378:metabolism; D018274:Electroporation / Q000379:methods*; D018014:Gene Transfer Techniques; D051379:Mice; D018485:Muscle Fibers, Skeletal / Q000166:cytology / Q000502:physiology*; D010957:Plasmids / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism
D011993:Recombinant Fusion Proteins; D004247:DNA