A steroid-inducible promoter for the controlled overexpression of cloned genes in eukaryotic cells.
Previous studies have shown that members of the steroid receptor family of transcriptional regulators can function synergistically when bound to multiple arrays of specific DNA binding sites known as hormone response elements, usually located upstream of target genes. We have constructed a mammalian expression vector containing a synthetic promoter composed of five high-affinity glucocorticoid response elements (termed GRE5) placed upstream of the adenovirus 2 major late promoter "TATA" region. In transiently transfected HeLa cells in the presence of dexamethasone, the GRE5 promoter was at least 50-fold more efficient than the mouse mammary tumor virus long terminal repeat in expressing bacterial chloramphenicol acetyltransferase activity. When the GRE5 vector was introduced stably into the HeLa cell genome, chloramphenicol acetyltransferase activity was induced from 10- to >50-fold by dexamethasone in six of eight responsive clones. The levels of both basal and induced expression varied from one clone to the next, probably due to an effect of chromosomal location on promoter activity. When propagated stably in HeLa cells in an Epstein-Barr virus episomal vector, the GRE5 promoter was > 50-fold inducible and its activity was strictly dependent on the presence of dexamethasone. We also show that the GRE5 promoter stably propagated in HeLa cells is inducible by progesterone in the presence of a transiently transfected progesterone receptor expression vector. The GRE5 promoter should be widely applicable for the strictly controlled high-level expression of target genes in eukaryotic cells that contain either the glucocorticoid or progesterone receptors.
Department of Biochemistry, McGill University, Montreal, PQ, Canada.
S
S
Mader
J H
JH
White
D000260:Adenoviruses, Human / Q000235:genetics*; D001665:Binding Sites; D015500:Chloramphenicol O-Acetyltransferase / Q000096:biosynthesis* / Q000235:genetics; D003001:Cloning, Molecular; D004247:DNA / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism*; D003907:Dexamethasone / Q000494:pharmacology*; D015870:Gene Expression; D005822:Genetic Vectors; D006367:HeLa Cells; D006801:Humans; D008324:Mammary Tumor Virus, Mouse / Q000235:genetics*; D011401:Promoter Regions, Genetic / Q000187:drug effects; D011965:Receptors, Glucocorticoid / Q000378:metabolism*; D011980:Receptors, Progesterone / Q000235:genetics / Q000378:metabolism*; D016385:TATA Box; D014162:Transfection
D004268:DNA-Binding Proteins; D011965:Receptors, Glucocorticoid; D011980:Receptors, Progesterone; D003907:Dexamethasone; D004247:DNA; D015500:Chloramphenicol O-Acetyltransferase