Figure 2

Transduction of primary rat skeletal myoblasts by the retroviral vector MLV-CX43-EGFP and resultant overexpression of connexin 43. Connexin 43 and EGFP were expressed from a single bicistronic transcription unit. (A) Transduction of primary rat skeletal myoblasts using various dilutions of the MLV-CX43-EGFP viral vector preparation. Medium in the wells of a 24-well plate containing non-confluent primary myoblasts was aspirated and the wells were filled with 1 ml of the non-diluted and diluted viral vector suspensions supplemented with Polybrene™ (10 μg/ml). After 48 hours the cells were trypsinised, washed in complete DMEM medium, resuspended in PBS and used for FACS analysis to determine the efficiency of transfection relying on EGFP expression. The obtained data were averaged among 3 wells for each virus vector dilution. (B) Concomitant use of RetroNectin^®-mediated viral vector concentration and polycation transduction enhancement for delivery of the connexin 43 and EGFP genes into primary skeletal myoblasts using MLV-CX43-EGFP vector. Transduction experiments were performed in 6-well plates containing non-confluent myoblasts in the presence of the polycation Polybrene™ (10 μg/ml), in the presence of the polycation Transfectam^® (5 μg/ml) or without addition of polycations. The efficiency of transduction was determined using the EGFP marker from the FACS subtraction plots using non-transduced primary skeletal myoblasts as a control, non-fluorescent cell population. The data were averaged among 6 individual transduction experiments. (C) Fractions of EGFP-positive cells in the population of connexin 43/EGFP transduced primary skeletal myoblasts during continuous passaging. The cells were split and re-seeded in the ratio 1:10 each week with concomitant FACS measurement of the fraction of the EGFP-expressing cells. (D) Results of FACS analysis of populations of primary skeletal myoblasts plotted as histograms (x-axis – intensity of green fluorescence, y-axis – counts of cells). Purple graph corresponds to non-transduced primary skeletal myoblasts, red graph corresponds to the primary skeletal myoblasts after RetroNectin^®-mediated Transfectam^®-enhanced transduction by MLV-CX43-EGFP, green graph represents primary skeletal myoblasts, which were preparatively sorted for EGFP expression after transduction by MLV-CX43-EGFP. (E) Western blotting analysis of connexin 43 expression in connexin 43 transduced skeletal myoblasts (after EGFP-based preparative sorting). The cell extracts were analysed by electrophoresis in a SDS-PAGE gel and immunoblotting using anti-connexin 43 antibody. Lane 1 corresponds to non-transduced primary skeletal myoblasts, lane 2 corresponds to connexin 43 transduced primary skeletal myoblasts, lane 3 corresponds to connexin 43 transduced primary skeletal myoblasts, which were passaged 13 weeks in tissue culture.