Lipofection of insulin-producing RINm5F cells: methodological improvements.
Cationic lipid/DNA-complexes have been widely used as gene transfer vectors because they are less toxic and immunogenic than viral vectors. The aim of the present study was to improve and characterize lipofection of an insulin-producing cell line. We compared the transfection efficiency of seven commercially available lipid formulations (Lipotaxi, SuperFect, Fugene, TransFast, Dosper, GenePORTER and LipofectAMINE) by flow cytometry analysis of GFP-expression. In addition, we have determined the influences of centrifugation, serum and a nuclear localization signal peptide on the lipofection efficiency. We observed that two lipid formulations, GenePORTER and LipofectAMINE, were able to promote efficient gene transfer in RINm5F cells. However, GenePORTER exhibited the important advantage of being able to transfect cells in the presence of serum and with less cytotoxicity than LipofectAMINE. LipofectAMINE-induced RINm5F cell death could partially be counteracted by TPA, forskolin or fumonisin beta(1). Finally, both centrifugation and a nuclear localization signal peptide increased transfection efficiency.
Barbu
Andreea
A
Department of Medical Cell Biology, Uppsala University.,Uppsala.,Sweden. Andreea.Barbu@medcellbiol.uu.se
Welsh
Nils
N
D000818:Animals; D002273:Carcinogens / Q000494:pharmacology; D016923:Cell Death / Q000187:drug effects; D002460:Cell Line; D005576:Colforsin / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D037341:Fumonisins / Q000494:pharmacology; D015870:Gene Expression; D005822:Genetic Vectors / Q000737:chemistry*; D049452:Green Fluorescent Proteins / Q000096:biosynthesis / Q000235:genetics; D007328:Insulin / Q000378:metabolism; D000078790:Insulin Secretion; D008055:Lipids / Q000737:chemistry*; D051381:Rats; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014162:Transfection / Q000379:methods*
D002273:Carcinogens; D004791:Enzyme Inhibitors; D037341:Fumonisins; D007328:Insulin; D008055:Lipids; D049452:Green Fluorescent Proteins; D005576:Colforsin; C056933:fumonisin B1; D013755:Tetradecanoylphorbol Acetate