Direct gene quantitation by PCR reveals differential accumulation of ectopic enzyme in rat-1 cells, v-fos transformants, and revertants.
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.
Bahramian
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MB
Division of Toxicology, Whitaker College of Health Science and Technology, Massachusetts Institute of Technology, Cambridge 02139, USA.
Zarbl
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D000818:Animals; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism; D002471:Cell Transformation, Neoplastic; D004247:DNA / Q000032:analysis / Q000302:isolation & purification / Q000378:metabolism; D018274:Electroporation; D019286:Endopeptidase K; D015870:Gene Expression; D016762:Genes, fos; D007700:Kinetics; D008979:Moloney murine leukemia virus; D010957:Plasmids; D016133:Polymerase Chain Reaction / Q000379:methods*; D011401:Promoter Regions, Genetic; D051381:Rats; D011994:Recombinant Proteins / Q000032:analysis / Q000096:biosynthesis; D012091:Repetitive Sequences, Nucleic Acid; D012697:Serine Endopeptidases; D014162:Transfection; D001616:beta-Galactosidase / Q000032:analysis / Q000096:biosynthesis*
D011994:Recombinant Proteins; D004247:DNA; D001616:beta-Galactosidase; D012697:Serine Endopeptidases; D019286:Endopeptidase K