Ventricular arrhythmias are a major cause of death following cardiac infarction. One possible therapy could be the transplantation of connexin 43 (Cx43) expressing cells (Roell et al. 2007). In order to design a feasible gene therapy approach with autologous cells we here demonstrate ex vivo viral-based gene transfer of Cx43 in embryonic and adult murine skeletal muscle cells. Skeletal myoblasts were harvested from embryonic or adult wild-type mice by enzymatic digestion of the diaphragm. For gene transfer a lentivirus was generated expressing Cx43 and EGFP under the ubiquitous CMV-promotor. Successful transduction of skeletal muscle cells was proven by EGFP fluorescence; Cx43 expression was further confirmed by immunostainings. Cultured skeletal myoblasts showed intact cell biological characteristics because they spontaneously fused forming elongated myotubes, which stained positive for the muscle-specific markers MyoD, Myogenin and Alpha-actinin. We assessed functional coupling between transduced cells by dye-transfer-studies. For this purpose single-cell-electroporation was performed with a high-current-amplifier and sharp electrodes filled with 40 mM KCl. Cell-membranes of single EGFP positive myotubes were permeabilized with an alternating current of 400Hz (20-50 nA; 30-180 sec) and the electroporated myotubes filled with two different dyes. Alexa350 (349 Da) passes through gap-junctions whereas dextran coupled Alexa546 (10 kDa) is a larger molecule and passes only through cytoplasmic bridges. Upon electroporation, we observed passage of Alexa350 in adjacent EGFP positive myotubes within 30 sec (n=29). Importantly, this was never observed in EGFP negative myotubes and Alexa546-dextran was restricted to the loaded cell. These results were identical in embryonic and adult skeletal muscle preparations. We conclude that virus-based Cx43 expression in skeletal muscle cells results in functional gap junction channels. Roell, W. et al. 2007. Nature 450: 819-24