Abstract
Activated estrogen receptor binds to specific genomic DNA termed estrogen response element (ERE) and enhances the transcription of target genes. There are numerous studies to examine ERE transcriptional activation in cell lines or tumor cells but little is known about primary culture cells especially in the pituitary. To determine the direct transcriptional activity of ERE in primary culture cells, we established the adenovirus-mediated reporter assay system for ERE transcriptional activation in lactotroph cells. 2×ERE, which was originally constructed and the most responsive for estrogenic induction among 1× to 4×ERE, was used for reporter DNA construct. Because the pituitary cell population consists of several cell types including estrogen-responsive gonadotroph cells, Cre-loxP system was used to localize the luciferase gene expression in lactotroph cells. Cre recombinase gene with nuclear localization signal was expressed under the control of prolactin promoter and the luciferase reporter gene was flanked by loxP sites. Double-fluorescence immunocytochemistry revealed that both Cre recombinase and luciferase proteins were expressed specifically in lactotroph cells when pituitary cells in primary culture were infected with two adenovirus vectors carrying these proteins. Treatment with 1 nM estradiol increased the ERE activity about 9-fold in the presence of dextran/charcoal-treated serum. This system will be useful to detect the ERE transcriptional activity in primary lactotroph cells. [J Physiol Sci. 2006;56 Suppl:S218]
