Direct Plasmid DNA Transfection into Differentiated Mouse C2C12 Myotubes

Abstract

Background: Models of muscle cell culture allow simple manipulation to optimize gene transfer experiments, offering a significant opportunity to model muscle effects by allowing precise control of the cell culture environment in a cost-effective manner. Often, gene transfer is performed in myoblasts, which are then differentiated into myotubes, a skeletal muscle model. In this study, we aimed to compare the efficiency of direct transfection of differentiated myotubes by chemical and physical plasmid methods. Methods: Differentiated myotubes were transfected with reporter plasmids. Transfection efficiency and cell death were quantified. Proinflammatory proteins are produced by cells and tissues after DNA transfection; therefore, the secretion of proinflammatory chemokines was quantified. Results: In this study, we demonstrate that C2C12 myotubes can be effectively transfected using multiple methods. While a chemical method did not cause cell death, myotubes were not efficiently transfected. Electroporation delivery produced a higher transfection efficiency coupled with higher cell death. A subset of proinflammatory chemokine proteins was secreted, which replicated secretion by skeletal muscle. Discussion: These results suggest that differentiated myotubes can be directly transfected invitro, which may represent a useful experimental model. However, more studies are needed to assess the value of this model in predicting invivo responses.

Keywords

• C2C12 myotubes
• plasmid DNA
• transfection
• electroporation
• chemical methods