Abstract
Background The aim of this study was to determine the optimal non-viral transfection method for use in human smooth muscle cells (SMC) and endothelial cells (EC).MethodsCoronary Artery (CoA) and Aortic (Ao) SMC and EC were transfected with a reporter plasmid, encoding chloramphenicol acetyltransferase type 1 (CAT), with seven different transfection reagents, two electroporation methods and a photochemical internalization (PCI) method. CAT determination provided information regarding transfection efficiency and total protein measurement was used to reflect the toxicity of each method.ResultsElectroporation via the nucleofector machine was the most effective method tested. It exhibited a 10 to 20 fold (for SMC and EC, respectively) increase in transfection efficiency in comparison to the lipofection method combined with acceptable toxicity. FuGene 6 and Lipofectamine PLUS were the preferred transfection reagents tested and resulted in 2 to 60 fold higher transfection efficiency in comparison to the PCI which was the least effective method.ConclusionThis study indicates that electroporation via the nucleofector machine is the preferred non-viral method for in vitro transfection of both human aortic and coronary artery SMC and EC. It may be very useful in gene expression studies in the field of vascular biology. Through improved gene transfer, non-viral transfer techniques may also play an increasingly important role in delivering genes to SMC and EC in relevant disease states.
Highlights
The aim of this study was to determine the optimal non-viral transfection method for use in human smooth muscle cells (SMC) and endothelial cells (EC)
In this study we present extensive investigations performed with transfection reagent mediated transfections, electroporation and photochemical internalization (PCI)
Our results showed that electroporation via the nucleofector machine turned out to be the most effective non-viral method for in vitro transfection of both human SMC and EC, while FuGene6 and Lipofectamine PLUS appeared as best performing lipofection reagents
Summary
The aim of this study was to determine the optimal non-viral transfection method for use in human smooth muscle cells (SMC) and endothelial cells (EC). Several methods have been described to introduce DNA expression vectors into mammalian cells in vitro and in vivo: calcium phosphate precipitation, microinjection, electroporation, receptor-mediated gene transfer, particle guns, viral vectors, and lipofection [1,2,3]. Viral vector systems, derived from modified animal or human viruses, resulting in replication-deficient vectors [4], represent a powerful transfection tool. Their immunogenicity, oncogenic properties, inactivation (page number not for citation purposes). The use of cationic liposome/DNA complexes (lipofection) for gene transfer into somatic cells has become a popular method of delivering genes. Transfection using lipofection offers some advantages over viral vectors, such as simplicity of production, low toxicity and low immunogenicity
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