Plasmid Transfection and Generation of Stable Cell Lines

Plasmid transfections were performed as described³. Briefly, primary myoblasts were cultured until they reached 50-60% confluency in the absence of antibiotics. Purified plasmid DNA was prepared by diluting 2 μg of DNA and transfected using Lipofectamine 2000 (Invitrogen). The transfection mixture was incubated for 3 hours at 37°C. Following incubation, 1 mL of antibiotic-free growth media was added, and cells were allowed to grow overnight at 37°C. The next day, the growth media was replaced with fresh antibiotic-free growth media, which was changed every 48 hours post-transfection. For antibiotic selection, G418 (Invitrogen) was added to the growth media at a concentration of 400 μg/mL. Transfected cultures were maintained and passaged to generate stable pooled cultures.