Akademik Veri Yönetim Sistemi
Yeşilkaya A. (Yürütücü), Tokay A.
TÜBİTAK Projesi, 2008 - 2009
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 RasN17 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 RasN17 DNA.
Conclusion: According to the difficulty of transfections of
primary cell culture, we used FuGENE reagent with different DNA and plasmid
ratio describes by the manufacturer and obtained better transfection efficiency
in primary cultured vascular smooth muscle cells. Our findings, precisely implicates
to use FuGENE reagent for to get a better transfection efficiency in primary
cultured cells, especially in primary cultured VSMCs.