Reexamination of the effect of endotoxin on cell proliferation and transfection efficiency

Plasmid DNA purified from bacterial cells can be contaminated with endotoxi n to different extents, depending on the purification method. Earlier repor ts indicate that endotoxin can decrease transfection efficiency in many euk aryotic cell lines; however the amount of endotoxin required for inhibition is unclear. We determined endotoxin effects in several cell lines and obse rved that endotoxin levels greater than or equal to 10 000 endotoxin units (EU) were needed to significantly affect cell proliferation and viability: levels greater than 2000 EU/mu g DNA were required to significantly inhibit transfection for all but one (Huh-7) of the cell lines tested. These endot oxin levels are significantly higher than endotoxin contamination in plasmi d DNA purified by anion exchange, CsCl2 gradient and endotoxin-free purific ation technology, but not as high as a crude alkaline lysis preparatory met hod. Plasmid DNA prepared using anion exchange technology was comparable to endotoxin-free technology in terms of transfection efficiency. Even Huh-7 cells, which are markedly more sensitive to endotoxins, have comparable tra nsfection efficiencies using plasmid DNA purified by either of these two me thods. We conclude that for those cell fines commonly used for transfection studies, endotoxin-free, quality DNA is not necessary because significantl y higher levels of bacterial endotoxins are required to inhibit either cell proliferation or transfection.