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.
Department of Internal Medicine, National Taiwan University Hospital and National Taiwan University College of Medicine, Taipei 100, Taiwan, Republic of China.
Jen-Feng
JF
Lai
Shin-Hun
SH
Juang
Yi-Mei
YM
Hung
Hsin-Yuan
HY
Cheng
Tzu-Ling
TL
Cheng
Keith E
KE
Mostov
Tzuu-Shuh
TS
Jou
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