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|Antigen/Gene or Protein Targets||Antioxidant Response Element (ARE)- Luciferase|
|Disease Keywords||Cancer; drug metabolism|
|Relevance||The stable human mammary MCF7-derived reporter cell line called AREc32 contains copies of the rat GST antioxidant response element (ARE) linked to a luciferase gene, such that the induction of the ARE results in luciferase activity. ARE is a transcriptional regulatory element involved in the activation of genes coding for a number of antioxidant proteins and enzymes that work in concert to protect tissues from oxidative insults. This cell line can be used to investigate whether anticancer drugs can induce ARE-driven gene expression.|
|Production Details||The ARE-luciferase reporter plasmid was generated using the pGL3-promoter vector containing an SV40 promoter upstream of the firefly luciferase gene. They differ in the number of copies of ARE sequences that have been inserted, in head-to-tail orientation, through Nhe I & Xho I restrictionsites upstream of the promoter-luc+ transcriptional unit. A plasmid was made containing eight copies of the ARE (5'-GTGACAAAGCA-3', with the minimal functional sequence underlined) present in both rat GSTA2 and mouse gsta1; called pGL-8xARE. A linker with the sequence of 5'-CCC-3' and 5'-GGG-3' on the opposite strand was placed between individual cis-elements.pGL-8xARE, was stably transfected into MCF7 cells using the calcium phosphate method. Transfected cells were selected using 0.8 mg/mL G418 in the media for 3 to 4 weeks. The G418-resistant clones were isolated and screened by measuring their basal and inducible (obtained by treatment with 50 Amol/L t-BHQ) luciferase activities as described above. Positive clones, which showed low background and high inducible luciferase activity, were passaged and maintained in growth medium containing 0.8 mg/mL G418.|
|Research Area||Cancer, Drug Discovery & Development|
|Recommended Growing Conditions||DMEM with glutamax supplemented with 10% fetal bovine serum and antibiotics. Do not culture beyond 15 passages after revival.|
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