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An ecdysone and tetracycline dual regulatory expression system for studies on Rac1 small GTPase-mediated signaling.
- Source :
- American Journal of Physiology: Cell Physiology; Sep2003, Vol. 285 Issue 3, pC711-C719, 9p, 12 Color Photographs, 16 Black and White Photographs, 20 Graphs
- Publication Year :
- 2003
-
Abstract
- 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. Important]y, 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. [ABSTRACT FROM AUTHOR]
- Subjects :
- ECDYSONE
TETRACYCLINE
GUANOSINE triphosphatase
GREEN fluorescent protein
Subjects
Details
- Language :
- English
- ISSN :
- 03636143
- Volume :
- 285
- Issue :
- 3
- Database :
- Complementary Index
- Journal :
- American Journal of Physiology: Cell Physiology
- Publication Type :
- Academic Journal
- Accession number :
- 10775647
- Full Text :
- https://doi.org/10.1152/ajpcell.00064.2003