title: Cell-type-specific modulation of PDGF-B regulatory elements via viral enhancer competition: a caveat for the use of reference plasmids in transient transfection assays.
abstract: The human platelet-derived growth factor-B (PDGF-B) gene has been shown to display a wide range of levels of mRNA transcription in a variety of cell types. Functional analyses of PDGF-B gene expression have begun to reveal intricate, cell-type-specific regulatory mechanisms involving multiple control elements. We have previously isolated and characterised several elements involved in the control of human PDGF-B gene expression in the JEG-3 choriocarcinoma cell line and in the breast cancer-derived cell line, ZR-75. Assessment of the positive or negative regulatory contributions of these elements was carried out using transient transfection assays. Such studies routinely require the inclusion of a reference plasmid in order to determine transfection efficiency. Here we show that competition for regulatory factors occurs in transfected cells between viral enhancers and elements regulating PDGF-B gene transcription. A frequently used reference plasmid which utilises the SV40 promoter and enhancer region to drive expression of a beta-galactosidase reporter gene was found to severely repress the activity of a co-transfected reporter construct containing the PDGF-B promoter and its intronic enhancer in JEG-3 cells. This competition was localised to the enhancer region of the SV40 regulatory sequences and surprisingly, the effect was reversed in ZR-75 cells; where increasing the amount of reference plasmid strongly stimulated the activity of the PDGF-B construct. These results imply that the same intronic region which functions equally well as an enhancer in two distinct cell-types, may operate in response to different transcription factor complements. Furthermore, this data demonstrates that the choice of reference plasmid and its quantitative use can be a crucial factor when examining putative regulatory elements by transient transfection methods.
authors.affiliation: Department of Animal Development and Genetics, University of Uppsala, Sweden.
authors.forename: G I
authors.initials: GI
authors.lastname: Adam
authors.forename: S J
authors.initials: SJ
authors.lastname: Miller
authors.forename: E
authors.initials: E
authors.lastname: Ullerås
authors.forename: G C
authors.initials: GC
authors.lastname: Franklin
mesh_terms: D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics; D004742:Enhancer Elements, Genetic; D005786:Gene Expression Regulation; D017930:Genes, Reporter; D006801:Humans; D010957:Plasmids; D010982:Platelet-Derived Growth Factor / Q000235:genetics*; D011401:Promoter Regions, Genetic; D011518:Proto-Oncogene Proteins / Q000235:genetics*; D020574:Proto-Oncogene Proteins c-sis; D013539:Simian virus 40 / Q000235:genetics*; D014162:Transfection; D014407:Tumor Cells, Cultured
chemical_list: D010982:Platelet-Derived Growth Factor; D011518:Proto-Oncogene Proteins; D020574:Proto-Oncogene Proteins c-sis; D015500:Chloramphenicol O-Acetyltransferase