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METHODS IN CELL PHYSIOLOGY

An ecdysone and tetracycline dual regulatory expression system for studies on Rac1 small GTPase-mediated signaling

¹Department of Internal Medicine, National Taiwan University Hospital and National Taiwan University College of Medicine, and ²Division of Cancer Research, National Health Research Institutes, Taipei 100, Taiwan, Republic of China; and ³Department of Anatomy and Cardiovascular Research Institute, University of California, San Francisco, California 94143

Submitted 19 February 2003 ; accepted in final form 3 May 2003

Regulated expression systems are invaluable for studying gene function, offer advantages of dosage-dependent and temporally defined gene expression, and limit possible clonal variation when toxic or pleiotropic genes are overexpressed. Previously, establishment of inducible expression systems, such as tetracycline- and ecdysone-inducible systems, required assessment of the inducible characteristics of individual clones by tedious luciferase assays. Taking advantage of a green fluorescent protein (GFP) reporter controlled by tetracycline- or ecdysone-responsive element and fluorescence-activated cell sorting, we propose a simple and efficient strategy to select highly inducible cell lines according to their fluorescence profiles after transiently transfecting the candidate cell pools with a surrogate GFP reporter. We have demonstrated that tetracycline- and ecdysone-inducible systems could be set up in Madin-Darby canine kidney and HEK-293 cells by employing this selection scheme. Importantly, this dual regulatory expression system is applied in studying the complex interplay between two Ras-related small GTPases, Cdc42 and Rac1, on detachment-induced apoptosis. Furthermore, establishment of two tightly regulated expression systems in one target cell line could be of great advantage for dissecting small GTPase Rac1-transduced signaling pathways by using global gene expression approaches such as proteomic assays.

fluorescence-activated cell sorting; green fluorescent protein; Ras small GTPases; anoikis

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