Lipopolysaccharide is a frequent contaminant of plasmid DNA preparations and can be toxic to primary human cells in the presence of adenovirus.
Endotoxin (lipopolysaccharide, LPS) is commonly found as a contaminant in plasmid DNA preparations. We demonstrate here that the quantities of LPS typically contaminating DNA preparations can generate a toxicity to primary cells (primary human skin fibroblasts, primary human melanoma cells) in the presence of entry-competent adenovirus particles. Toxicity can be observed with as little as 100 ng/ml free LPS or 100 pg/ml LPS when the LPS is assembled into polylysine/adenovirus complexes. Simple and effective methods of removing the contaminating LPS using either a polymyxin B resin or Triton X-114 extraction are described. Treatment of DNA samples to remove LPS eliminates the toxicity to primary cells.
Cotten
M
M
IMP, Research Institute of Molecular Pathology, Vienna, Austria.
Baker
A
A
Saltik
M
M
Wagner
E
E
Buschle
M
M
D000256:Adenoviridae / Q000235:genetics*; D002478:Cells, Cultured; D002499:Centrifugation, Density Gradient; D004247:DNA / Q000302:isolation & purification; D004340:Drug Contamination; D006801:Humans; D008070:Lipopolysaccharides / Q000302:isolation & purification / Q000633:toxicity*; D010957:Plasmids / Q000302:isolation & purification*; D011107:Polylysine; D014162:Transfection; D014407:Tumor Cells, Cultured
D008070:Lipopolysaccharides; D011107:Polylysine; D004247:DNA