Title: Direct gene quantitation by PCR reveals differential accumulation of ectopic enzyme in rat-1 cells, v-fos transformants, and revertants.
Authors: Bahramian, M B; Zarbl, H
Published In PCR Methods Appl, (1994 Dec)
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.
PubMed ID: 7580898
MeSH Terms: Animals; Cell Line; Cell Nucleus/metabolism; Cell Transformation, Neoplastic*; DNA/analysis; DNA/isolation & purification; DNA/metabolism; Electroporation; Endopeptidase K; Gene Expression; Genes, fos*; Kinetics; Moloney murine leukemia virus; Plasmids; Polymerase Chain Reaction/methods*; Promoter Regions (Genetics)*; Rats; Recombinant Proteins/analysis; Recombinant Proteins/biosynthesis; Repetitive Sequences, Nucleic Acid; Serine Endopeptidases; Transfection; beta-Galactosidase/analysis; beta-Galactosidase/biosynthesis*