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GENERATION OF TWO NOVEL CELL LINES THAT STABLY EXPRESS HAR AND FIREFLY LUCIFERASE GENES FOR ENDOCRINE SCREENING
Citation:
Bobseine, K L., W. R. Kelce, P C. Hartig, AND L E. Gray. GENERATION OF TWO NOVEL CELL LINES THAT STABLY EXPRESS HAR AND FIREFLY LUCIFERASE GENES FOR ENDOCRINE SCREENING. Presented at Society for the Study of Reproduction, Madison, WI, July 15-18, 2000.
Description:
Generation of Two Novel Cell Lines that Stably Express hAR 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 AR 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 CHO (Chinese Hamster Ovarian) parental cells and two plasmids. The receptor plasmid was created to express hAR and confer zeomicin-resistence. The reporter plasmid was created to express firefly luciferase under the control of the MMTV (mouse mammary tumor virus) promotor and to confer neomicin-resistence. The MMTV promotor contains sequences homologous to the hAR and hGR response elements. The CHO cells were co-transfected using a standard calcium phosphate transfection technique with an hAR-zeomicin plasmid construct and a MMTV-luciferase-neomicin plasmid construct. Cells were grown in selection media containing both gentamicin and zeocin. A second stable line was created using the MDA-MB-453 cells, a human breast cancer line which contains endogenous AR and GR. After transfection with Fugene 6 using the manufacturer's protocol and the MMTV-luciferase-neomicin reporter plasmid alone, these cells were grown in selection media with gentamicin only. Resulting colonies from the CHO and MDA transfections were isolated and expanded. Colonies were screened for detectable AR/GR induction with the AR agonist, dihydroxytestosterone (DHT); the GR agonist, dexamethasone; and with the AR antagonist, hydoxyflutamide using a luciferase assay in 96-well plates. Both cell lines respond consistently to 0.1nM DHT induction over almost 100 passages. (This does not reflect EPA policy.)