Effect of DNA Contaminants on Calcium Phosphate-Based DNA Delivery and Gene Expression

Plasmid DNA is widely used to deliver genes into mammalian cells for the construction of new cell lines, gene therapy and gene expression studies. During DNA preparation, various contaminants can be introduced and reduce its delivery efficiency and create mutations that decrease the expression level of the delivered genes. We evaluated the effect of different plasmid DNA contaminants on calcium phosphate-based transfection efficiency as well as gene expression in Chinese hamster ovary cells. pCMVβ was transfected into the cells after spiking with five different contaminants conditions: ethanol, endotoxin, cesium chloride, ethidium bromide and the combination of the latter two contaminants. Transfection efficiencies were determined through the counting of the Lac-Z-expressing cells as well as quantitative PCR. The reversibility of the contaminant interaction with DNA was examined through cleaning of the spiked DNA followed by transfection. As concluded from our results, qPCR offers accurate measurement of transfection efficiency than the conventional Lac-Z activity assay. In general, all of the examined contaminants, with the exception of ethanol, have a significant negative effect on gene expression. While this effect is resulted from the reduced delivery in most of the used contaminants, ethidium bromide showed no significant decrease in delivery, indicating alternative mechanism for its negative effect on gene expression.