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293/CRE-Luciferase Host Cell Line Technical Manual No. TM0426 I II III IV I. Version 10132010 Introduction…….……………………………………………………………………………. Background…………………………………………………………………………………. Representative Data……………………………………………………………………… References ………………………………………………………………………………. Limited Use License Agreement………………………………………………………… 1 1 2 2 3 Introduction Catalog Number: M00188 Cell Line Name: 293/CRE-Luc Host Cell: 293 Quantity: Two vials of frozen cells (3×106 per vial) Stability: 16 passages Applications: Functional assays for Gs protein coupled receptors Freeze Medium: 45% culture medium, 45% FBS, 10% DMSO Culture Medium: DMEM, 10% FBS, 100 µg/ml Hygromycin B, 250 µg/ml G418 Mycoplasma Status: Negative Storage: Liquid nitrogen immediately upon delivery II. Background The G-protein-coupled receptor (GPCR) superfamily represents one of the most important classes of drug targets in the industry. At present, about 50% of all medicines currently in the market are targeted to GPCRs. GPCRs can be divided into several subfamilies according to the kind of Gα protein to which they couple: There are Gαs-, Gαi-, and Gαq-coupled receptors. Gαs proteins can activate adenyl cyclase and increase the intracellular concentration of cAMP. At GenScript, we employ both 3xCRE element and firefly luciferase as reporters to establish host cell lines that can be used for research involving GPCRs, particularly Gs-coupled GPCRs. This cell line demonstrates an approximately 50-fold dynamic range in the luciferase assay, providing a good screening window that is suitable for screening GPCR agonists and antagonists. -1- III. Representative Data CRE Inducibility of 293/CRE/Luc Cell Line: Unstimulated Control Stimulated (forskolin) 12829 699503 1 54 Background subtracted luciferase readout (RLU) Fold induction 293/CRE-Luc 800000 54-Fold RLU 600000 400000 200000 0 Forskolin 10M Cell Control Figure: Luciferase activities were monitored with Analyst HT plate reader upon treatment with forskolin. 293/CRE-Luc cell line was treated with forskolin as indicated. Data represent means ± SEM for duplicate samples. IV. References 1. Conway, S., et al., Characterisation of human melatonin MT (1) and MT (2) receptors by CRE-luciferase reporter assay. Eur J Pharmacol, 2000. 390(1-2): p. 15-24. 2. Klabunde, T. (2002) Drug design strategies for targeting G-protein-coupled receptors. Chembiochem. 3, 924– 44. GenScript USA Inc. 860 Centennial Ave., Piscataway, NJ 08854 Tel: 732-885-9188, 732-885-9688 Fax: 732-210-0262, 732-885-5878 Email: [email protected] Web: http://www.genscript.com For Research Use Only. -2- Limited Use License Agreement This is a legal agreement between you (Licensee) and GenScript USA Inc. governing use of GenScript's stable cell line products and protocols provided to licensee. By purchasing and using the stable cell line, the buyer agrees to comply with the following terms and conditions of this label license and recognizes and agrees to such restrictions: 1) 2) 3) The products are not transferable and will be used at the site where they were purchased. Transfer to another site owned by buyer will be permitted only upon written request by buyer followed by subsequent written approval by GenScript. The purchaser cannot sell or otherwise transfer (a) this product (b) its components or (c) materials made using this product or its components to a third party. The products sold by GenScript are for laboratory and animal research purposes only. The products are not to be used on humans, for consumption, or for any unlawful uses. GenScript USA Inc. will not assert against the buyer a claim of infringement of patents owned or controlled by GenScript USA Inc. and claiming this product based upon the manufacture, use or sale of a clinical diagnostic, therapeutic and vaccine, or prophylactic product developed in research by the buyer in which this product or its components has been employed, provided that neither this product nor any of its components was used in the manufacture of such product. For information on the use of this product for other purposes, contact Marketing Department, GenScript USA Inc., 860 Centennial Avenue, Piscataway, New Jersey 08840, U.S.A. Phone: 1-732885-9188. Fax: 1-732-210-0262. Email: [email protected]. -3-