Discordant estimates of heterologous promoter activity as determined by reporter gene mRNA levels and enzyme activity.
In this study, the human cytomegalovirus (CMV) promoter fused to the lacZ (beta-gal) reporter gene was transfected into neuroblastoma SK-N-BE(2)-C cells, and phorbol ester-stimulated promoter activity assessed by both PCR quantitation of reporter gene mRNA levels and enzyme activity. Surprisingly, significant differences were observed in the induction profile of CMV promoter activity as judged by these two independent methods of analysis. For example, at 24 hrs post-transfection beta-gal activity was elevated 7.3-fold in phorbol ester-treated cells, whereas 2.4-fold increases were observed in the cognate mRNA levels. These findings demonstrate the efficacy of quantitative PCR methodology to evaluate promoter activity in DNA-mediated cell transfection analyses, and raise a cautionary note on the reliance of reporter gene enzyme activity to estimate the transcriptional activity of heterologous promoters.
Kovacs
D M
DM
Department of Psychiatry, University of Pittsburgh School of Medicine, PA 15213.
Kaplan
B B
BB
D001483:Base Sequence; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics* / Q000378:metabolism*; D003001:Cloning, Molecular; D003587:Cytomegalovirus / Q000235:genetics*; D005798:Genes, Bacterial; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D009447:Neuroblastoma; D009838:Oligodeoxyribonucleotides; D010957:Plasmids; D016133:Polymerase Chain Reaction / Q000379:methods; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism*; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D013997:Time Factors; D014162:Transfection; D014407:Tumor Cells, Cultured; D001616:beta-Galactosidase / Q000235:genetics* / Q000378:metabolism*
D009838:Oligodeoxyribonucleotides; D012333:RNA, Messenger; D011994:Recombinant Proteins; D015500:Chloramphenicol O-Acetyltransferase; D001616:beta-Galactosidase; D013755:Tetradecanoylphorbol Acetate