Observing better transfection efficiency in primary cultured VSMCs Comparison and development of two different protocols

OpenAlex İngilizce

Aim: In this study, we have evaluated the procedure of transient transfection efficiency of vascular smooth muscle cells (VSMCs) with Lipofectamine (Life Technologies) and FuGENE (ROCHE, FuGENE HD Reagent) transfection reagents using the pCH110 eukaryotic assay vector containing the lacZ reporter gene. We also did try these attainments to transfect VSMCs with Ras N17 DNA and affirmed our findings. Material Methods: Plasmid pcH110, which has been purified by cesium chloride gradient centrifugation, was used in all transfections as the assay vector. And c-H-Ras was observed via Western-blot technique. Results: Briefly, the transfection with FuGENE has been given the best results, comparing with Lipofectamin. Under our culture conditions for VSMCs, FuGENE transfection efficiency could be augmented by simply increasing the amount of plasmid DNA 1.5–3 times above the recommended concentration without any visible cytotoxicity. With the FuGENE reagent, optimal transfection efficiency was obtained for primary culture of VSMCs within the recommended concentrations, but at the top of the range. The results indicate that optimization of the transfection process should include plasmid DNA concentrations above the levels suggested by the manufacturers, in order to accomplish the highest transfection efficiency. And, these finding was also supported with our Western-blot results when VSMCs have been transiently transfected with Ras N17 DNA.