Efficient lipid-mediated transfection of DNA into primary rat hepatocytes.
Cationic lipids are an effective means for transfecting nucleic acids into a variety of cell types. Very few of these lipids, however, have been reported to be effective with primary cells. We report on the efficacy of several commercially available cationic lipid reagents to transfect plasmid DNA into primary rat hepatocytes in culture. The reagents tested in this study include TransfectAce, LipofectAmine, Lipofectin, N-[1-(2,3-dioleyloxy)propyl]-n,n,n-trimethylammoniumchloride (DOTMA), (N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethyl-ammonium methylsulfate (DOTAP), and cetyltrimethyl-ammonium bromide/dioleoylphosphatidylethanol-amine (CTAB/DOPE). Electron micrographic (EM) studies indicate that similar size Lipofectin and DOTAP vesicles contain DNA-like material internally and that these vesicles attach to the cell membrane. DOTAP vesicles are multilamellar, appear as clusters, and have a high DNA-to-lipid ratio. Lipofectin vesicles appear to attach to the cell surface as individual vesicles. The EM observations are consistent with current theories on the mechanism of transfection by cationic lipids. While Lipofectin has proven to be effective in transfection studies of primary cells in culture, we have found DOTAP to be a viable alternative. DOTAP yields transfection rates in hepatocytes comparable to DOTMA and Lipofectin, however, at lower concentrations of reagent and at considerably less cost. Optimal conditions for transfecting 5 micrograms of plasmid DNA with DOTAP were achieved by utilizing multilamellar (vortexed) vesicles at a concentration of 15 micrograms DOTAP per 2 ml media in 60-mm plates for 2 h transfection time. In this study, DOTAP has proven to be economical, easy to prepare, and very effective in transfecting DNA into primary rat hepatocytes.
Holmen
S L
SL
Department of Biological Sciences, Western Michigan University, Kalamazoo 49008, USA.
Vanbrocklin
M W
MW
Eversole
R R
RR
Stapleton
S R
SR
Ginsberg
L C
LC
D000818:Animals; D002478:Cells, Cultured; D005229:Fatty Acids, Monounsaturated; D008055:Lipids; D008081:Liposomes; D008099:Liver / Q000166:cytology* / Q000378:metabolism* / Q000648:ultrastructure; D008297:Male; D010714:Phosphatidylethanolamines; D010957:Plasmids; D000644:Quaternary Ammonium Compounds; D051381:Rats; D011916:Rats, Inbred F344; D013997:Time Factors; D014162:Transfection / Q000379:methods*
D005229:Fatty Acids, Monounsaturated; D008055:Lipids; D008081:Liposomes; D010714:Phosphatidylethanolamines; D000644:Quaternary Ammonium Compounds; C054005:N-(1-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium; C100004:transfectACE; C020888:1,2-dielaidoylphosphatidylethanolamine; C070046:1,2-dioleoyloxy-3-(trimethylammonium)propane