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

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.