Short technical reports. Effects of lipopolysaccharide on transfection efficiency in eukaryotic cells.
The suitability of different purification methods for preparation of plasmid DNA for transfection into eukaryotic cells was systematically investigated. The reporter plasmid, pRSVcat, was prepared using several methods, and residual impurities in the preparations were quantitated. Transfection with these preparations was performed with several cell lines (HeLa, Huh7, COS7 and LMH) and two transfection methods: liposome-mediated and calcium phosphate transfection. Transfection efficiencies were determined by measuring chloramphenicol acetyltransferase expression. Higher transfection efficiencies were obtained with plasmid preparations of higher purity (those prepared by anion-exchange chromatography or two rounds of CsCl-gradient centrifugation) than with preparations of lower purity (those prepared using a silica-based DNA adsorption method or a single round of CsCl centrifugation). The results also demonstrated specifically that increasing concentrations of lipopolysaccharides in plasmid preparations directly correlate with decreasing transfection efficiencies.
Weber
M
M
QIAGEN GmbH, Hilden, Germany.
Möller
K
K
Welzeck
M
M
Schorr
J
J
D000327:Adsorption; D002130:Calcium Phosphates; D002460:Cell Line; D002499:Centrifugation, Density Gradient; D015500:Chloramphenicol O-Acetyltransferase; D002852:Chromatography, Ion Exchange; D005057:Eukaryotic Cells; D015870:Gene Expression; D008070:Lipopolysaccharides / Q000494:pharmacology*; D008081:Liposomes; D010957:Plasmids / Q000302:isolation & purification; D014162:Transfection / Q000379:methods*
D002130:Calcium Phosphates; D008070:Lipopolysaccharides; D008081:Liposomes; D015500:Chloramphenicol O-Acetyltransferase