Expression of beta-galactosidase gene and endothelial nitric oxide synthase gene in rat vascular smooth muscle cells after in vitro lipotransfection.
The aim of this study was to optimize the conditions for in vitro lipotransfection of rat vascular smooth muscle cells (VSMC) with bacterial beta-galactosidase gene and bovine endothelial nitric oxide synthase (ecNOS) gene. Transfection efficiency of four liposomes: Transfectam, Lipofectin, Unifectin-10, and Maxifectin was compared. The best results (efficiency 1-5%) were obtained with Maxifectin, when transfections were performed in VSMC cultures being at 50% confluency, with 1 microg DNA and 10 microl liposome per well, and when the liposome/DNA complexes were coincubated with the cells for 24 h. This method allowed detection of the transgene activity 12 h after the beginning of the transfection, with maximum values between the second and fourth days. The expression of the potentially therapeutic ecNOS gene was evidenced by confirmation of ecNOS mRNA generation, indirect detection of active ecNOS protein and by measurement of nitrite ion accumulation in the medium from the transfected cell cultures.
Józkowicz
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A
Department of Clinical Biochemistry, Collegium Medicum of Jagiellonian University, Kopernika 15A, 31-501, Kraków, Poland. almar@kki.krakow.pl
Dulak
J
J
Guevara
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I
Wybrańska
I
I
Dembińska-Kieć
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A
D000818:Animals; D001013:Aorta, Thoracic / Q000166:cytology / Q000201:enzymology; D002417:Cattle; D003470:Culture Media; D008058:Dihydrolipoamide Dehydrogenase / Q000378:metabolism; D015971:Gene Expression Regulation, Enzymologic; D009131:Muscle, Smooth, Vascular / Q000166:cytology / Q000201:enzymology*; D019001:Nitric Oxide Synthase / Q000235:genetics*; D052250:Nitric Oxide Synthase Type III; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D051381:Rats; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014162:Transfection / Q000379:methods*; D001616:beta-Galactosidase / Q000235:genetics*
D003470:Culture Media; D012333:RNA, Messenger; D019001:Nitric Oxide Synthase; D052250:Nitric Oxide Synthase Type III; C496316:Nos3 protein, rat; D008058:Dihydrolipoamide Dehydrogenase; D001616:beta-Galactosidase