Human estrogen receptor(alpha) activity on estrogen responsive, integrated chloramphenicol acetyltransferase (CAT) reporter constructs in Chinese Hamster Ovary (CHO) cells.

摘要

The expression of integrated estrogen responsive reporter gene constructs was investigated by constructing stably transfected cell lines of Chinese Hamster Ovary (CHO-K1) cells with three estrogen responsive chloramphenicol acetyltransferase (CAT) reporter gene constructs. Using neomycin (G418) selection and a novel chloramphenicol selection, I obtained three types of integrated estrogen responsive promoter cell lines. The first type of estrogen responsive promoter contained two consensus estrogen response elements (2ERETATACAT). The second was a related estrogen regulated thymidine kinase promoter (2ERETKCAT), and the third was a natural promoter, Xenopus laevis vitellogenin B1 (VITCAT). Transcription by the estrogen receptor (ER) was measured by assay of the enzymatic activity of the protein produced from the integrated CAT cDNA. I determined the transcriptional activity of transfected human ER (hER) mutants on the integrated promoters from homogenous populations of cells transfected by liposomes with mutant ER expression vectors. To obtain a homogenous population of transfected cells, I used a magnetic bead selection technique.;In at least one cell stably transfected cell line, A6H10, there was constitutive expression of the reporter gene from the integrated 2ERETATACAT. The local genomic environment was able to activate this synthetic promoter since functionally inactive mutants of the hER were unable to suppress the constitutive activity of the A6H10 cell line. Constitutive expression of CAT from the 2ERETATACAT promoter introduced into A6H10 cells by transient transfection was not observed.;Two other 2ERETATACAT cell lines were transfected with an hER expression vector or mutant hER expression vectors. In these two cell lines, neither activation function one (AF1) nor activation function two (AF2) of hER;The estrogen receptors endogenous to CHO cells did not induce transcription of chromosomal copies of the more complex 2ERETKCAT promoter. In transient transfections hER