Rapid selection of tetracycline-controlled inducible cell lines using a green fluorescent-transactivator fusion protein.
We describe a modification of the tetracycline-controlled expression system that facilitates the rapid identification of tetracycline-sensitive clones. The TetR/VP16 transactivator protein was tagged with the green fluorescent protein (GFP) at its N-terminus. This results in a functional transactivator which allows cells expressing high levels of the modified transactivator to be selected by fluorescent-activated cell sorting. After expansion, single cell clones that maintain a high level of GFP fluorescence can be tested for their ability to transactivate a luciferase gene under control of the Tet operator, leading to the rapid identification of clones with strong inducibility.
Callus
B A
BA
Department of Pediatric Oncology, Dana-Farber Cancer Institute, 44 Binney Street, Boston, Massachusetts, 02115, USA. Callus@dfci.harvard.edu
Mathey-Prevot
B
B
D000818:Animals; D002460:Cell Line; D002469:Cell Separation; D002999:Clone Cells; D004318:Doxycycline / Q000494:pharmacology; D005434:Flow Cytometry; D015870:Gene Expression / Q000187:drug effects*; D017930:Genes, Reporter / Q000235:genetics; D049452:Green Fluorescent Proteins; D017405:Herpes Simplex Virus Protein Vmw65 / Q000235:genetics / Q000378:metabolism; D008156:Luciferases / Q000235:genetics / Q000378:metabolism; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D011401:Promoter Regions, Genetic / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism*; D013752:Tetracycline / Q000378:metabolism / Q000494:pharmacology*; D013997:Time Factors; D015534:Trans-Activators / Q000235:genetics / Q000378:metabolism*; D014162:Transfection
D017405:Herpes Simplex Virus Protein Vmw65; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D015534:Trans-Activators; D049452:Green Fluorescent Proteins; D008156:Luciferases; D013752:Tetracycline; D004318:Doxycycline