Optimization of cationic lipid-mediated gene transfer to airway epithelia.
The use of cationic lipids for gene transfer to airway epithelia has shown promise in in vitro and in vivo studies. However, previous studies have used a wide variety of different lipid preparations and different formulations. Few studies have been designed to optimize the variables involved in transfection, and none have been focused on airway epithelia. Therefore we examined variables that affect cationic lipid-mediated transfection of HeLa cells and of airway epithelial cells grown on permeable filter supports at the air-liquid interface. To quantitate expression of cDNA, we assayed expression of luciferase. We found that the ratio of DNA to lipid was an important variable that determined transfection efficiency. In both HeLa and airway epithelial cells, the optimum charge ratio of cationic lipid to anionic DNA was approximately 1.25, consistent with the notion that a positively charged complex facilitates interaction with the negatively charged cell membrane. After testing a series of readily available cationic lipids, we found that 1,2-dimyristyloxypropyl-N,N-dimethyl-hydroxyethyl ammonium bromide (DMRIE)/dioleoyl phosphatidylethandamine (DOPE) appeared to show good efficacy. The concentration of DNA and cationic lipid also played an important role: in HeLa cells the optimum concentration of cationic lipid was approximately 5 microM and in airway epithelial cells was approximately 15 microM.(ABSTRACT TRUNCATED AT 250 WORDS)
Fasbender
A J
AJ
Howard Hughes Medical Institute, Department of Internal Medicine, University of Iowa College of Medicine, Iowa City 52242, USA.
Zabner
J
J
Welsh
M J
MJ
D002412:Cations / Q000378:metabolism*; D004247:DNA / Q000378:metabolism; D004847:Epithelial Cells; D004848:Epithelium / Q000502:physiology; D018014:Gene Transfer Techniques; D006367:HeLa Cells; D006801:Humans; D008055:Lipids / Q000502:physiology*; D008156:Luciferases / Q000378:metabolism; D009227:Myristic Acids / Q000494:pharmacology; D010714:Phosphatidylethanolamines / Q000494:pharmacology; D000644:Quaternary Ammonium Compounds / Q000494:pharmacology; D014132:Trachea / Q000166:cytology / Q000502:physiology*; D014162:Transfection
D002412:Cations; D008055:Lipids; D009227:Myristic Acids; D010714:Phosphatidylethanolamines; D000644:Quaternary Ammonium Compounds; C087276:(3-dimyristyloxypropyl)(dimethyl)(hydroxyethyl)ammonium; C094877:dioleoyl phosphatidylethanolamine; D004247:DNA; D008156:Luciferases