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Direct gene quantitation by PCR reveals differential accumulation of ectopic enzyme in rat-1 cells, v-fos transformants, and revertants.

Division of Toxicology, Whitaker College of Health Science and Technology, Massachusetts Institute of Technology, Cambridge 02139, USA.

Abstract

Valid comparisons of gene promoter activities between different cell lines, and within a cell line, critically depend on accurate measurements of the number of genes introduced into the nuclei of cells. We have developed a simple method that allows direct and accurate quantitation of transfected plasmid DNA in cultured cells. The transfected DNA present in nuclei is copurified with genomic DNA without using phenol/chloroform extractions. DNA is amplified by PCR, and the amount of transfected DNA is read directly from a standard curve. By using the procedures described in this report, we have studied the relative expression of the Escherichia coli beta-galactosidase gene, driven by the wild-type Mo-MuLV LTR, in Rat-1 fibroblasts, FBJ v-fos-transformed Rat-1 (1302), and a revertant of v-fos-transformant (EMS-1-19) cell lines. The relative levels of expression of the transgene at 22 hr post-transfection in these three cell lines were 1:4:1, respectively, and at 48 hr post-transfection the respective ratios were 1:10.6:4. These results have significant implications for the use of cotransfected internal control plasmids to normalize data from transient transfection experiments to study promoter activities among different cell lines.

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