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GENERATION OF TWO STABLE CELL LINES THAT EXPRESS HER-ALPHA OR HER-ALPHA AND -BETA AND FIREFLY LUCIFERASE GENES FOR ENDOCRINE SCREENING
Citation:
Bobseine, K L., W. R. Kelce, P C. Hartig, AND L E. Gray Jr. GENERATION OF TWO STABLE CELL LINES THAT EXPRESS HER-ALPHA OR HER-ALPHA AND -BETA AND FIREFLY LUCIFERASE GENES FOR ENDOCRINE SCREENING. Presented at Society of Toxicology, San Francisco, CA, March 25 - 29, 2001.
Description:
Generation of Two Stable Cell Lines that Express hERa or
hERa and b and Firefly Luciferase Genes for Endocrine Screening
K.L. Bobseine*1, W.R. Kelce2, P.C. Hartig*1, and L.E. Gray, Jr.1
1USEPA, NHEERL, Reproductive Toxicology Division, RTP, NC, 2Searle, Reproductive Toxicology Division, Skokie, IL
The goal of this project was to develop stable cell lines which are able to detect ER agonists and antagonists for use in high-throughput in vitro screening assays. Two different cell lines were developed for this purpose, both utilizing the firefly luciferase reporter gene. The first stable line was created using T47D parental cells (human breast cancer cells), which express both hER a and b. The second stable cell line was created using ZR-75-1 parental cells (human breast cancer cells) which express only ER a. The reporter plasmid for both was designed to express firefly luciferase under the control of the ER response element (ERE-luc) and to confer neomicin-resistence. The cells were transfected with Fugene 6 using the manufacturer's protocol and the reporter plasmid construct. These cells were grown in selection media containing gentamicin at a concentration previously determined to be lethal to non-transfected cells. Resulting colonies from transfections were isolated and expanded. Colonies were screened for detectable ER/GR induction with the ER agonist, 17-b estradiol (E2); the GR agonist, dexamethasone; and with the ER antagonist, ICI, using a luciferase assay in 96-well plates. Both cell lines demonstrate luciferase induction by E2 exposure while ICI consistently inhibited estradiol-mediated luciferase induction. (This abstract does not necessarily reflect EPA policy.)