DESIGN OF OESTROGEN RESPONSIVE REPORTER CONSTRUCTS FOR USE IN CELL CULTURE: USE OF CMV PROMOTER
K Derecka, C-K Wang, ELR Sheldrick & APF Flint
Hormone-responsive reporter plasmids are widely used for the analysis of endocrine responses in target cells. We have investigated the effect of different promoters on reporter gene expression by oestrogen-responsive reporter constructs.
Plasmid constructs were transfected into an oestrogen responsive bovine endometrial stromal (BST) cell line. Two reporter gene products were analysed: secreted alkaline phosphatase(SeAP) and beta galactosidase.
When the vitellogenin consensus minimal oestrogen response element (ERE, 5'-GGTCAGAGTGACC-3') and the CMV immediate early promoter/enhancer were inserted into pSeAP basic, SeAP expression was inhibited by oestradiol (1 or 10 nM) during 48 hr culture; basal SeAP levels were 1400 ng per mg protein, reduced to 600 ng per mg in presence of 10nM oestradiol. The effect of oestradiol was blocked by the antioestrogen ICI 182,780 (500 nM). Addition of 5' and 3' nucleotides to the ERE -the optimum ERE sequence- (CAGGTCAGAGTGACCTG) did not alter the response.
Lack of oestradiol induced up-regulation of reporter gene was also observed with another reporter system, pCMVbeta. CMV driven expression of beta galactosidase was inhibited by presence of ERE upstream of reporter gene with no effect on CMV promoter beta galactosidase construct.
However, after insertion of SeAP under control of the thymidine kinase promoter in pBL plasmid (in the absence of the CMV promoter), basal levels of SeAP (40 ng per mg protein) were increased over 10-fold by 10nM oestradiol, and this effect was blocked by ICI 182,780. Lowest concentration of oestradiol found to increase expression was 0.01 nM.
The CMV promoter is widely used due to its reliability and compatibility with many cell lines. Our data show that presence of the CMV together with ERE in reporter constructs renders them unresponsive to oestradiol activation.