An ecdysone and tetracycline dual regulatory expression system for studies on Rac1 small GTPase-mediated signaling.
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.
Lai
Jen-Feng
JF
Department of Internal Medicine, National Taiwan University Hospital and National Taiwan University College of Medicine, Taipei 100, Taiwan, Republic of China.
Juang
Shin-Hun
SH
Hung
Yi-Mei
YM
Cheng
Hsin-Yuan
HY
Cheng
Tzu-Ling
TL
Mostov
Keith E
KE
Jou
Tzuu-Shuh
TS
D000818:Animals; D004440:Ecdysone / Q000494:pharmacology*; D005434:Flow Cytometry; D005786:Gene Expression Regulation / Q000187:drug effects; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins; D006801:Humans; D007202:Indicators and Reagents / Q000378:metabolism; D007668:Kidney / Q000166:cytology; D008164:Luminescent Proteins / Q000235:genetics; D008967:Molecular Biology / Q000379:methods*; D040901:Proteomics; D015398:Signal Transduction / Q000235:genetics; D013754:Tetracyclines / Q000494:pharmacology*; D020830:rac1 GTP-Binding Protein / Q000235:genetics* / Q000378:metabolism
D007202:Indicators and Reagents; D008164:Luminescent Proteins; D013754:Tetracyclines; D049452:Green Fluorescent Proteins; D004440:Ecdysone; D020830:rac1 GTP-Binding Protein