Stable luciferase reporter cell lines for signal transduction pathway readout using GAL4 fusion transactivators
While GAL4 fusion activators have been widely used for dissecting signal transduction pathways in transient assays, there has been surprisingly little reported on utilizing cell lines with stably integrated fusion activators. To avoid problems with the efficiency and reproducibility inherent to tran...
| Veröffentlicht in: | BioTechniques. - 1993. - 30(2001), 5 vom: 01. Mai, Seite 1134-8, 1140 |
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| Format: | Aufsatz |
| Sprache: | English |
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2001
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| Zugriff auf das übergeordnete Werk: | BioTechniques |
| Schlagworte: | Journal Article Cyclic AMP Response Element-Binding Protein DNA-Binding Proteins Fungal Proteins GAL4 protein, S cerevisiae Recombinant Fusion Proteins Saccharomyces cerevisiae Proteins Trans-Activators Transcription Factors Epidermal Growth Factor mehr... |
| Zusammenfassung: | While GAL4 fusion activators have been widely used for dissecting signal transduction pathways in transient assays, there has been surprisingly little reported on utilizing cell lines with stably integrated fusion activators. To avoid problems with the efficiency and reproducibility inherent to transient transfection, we describe here the generation and characterization of HeLa reporter cell lines, which contain a stably integrated luciferase gene responsive to stably integrated and constitutively expressed GAL4-CREB or GAL4-Elk1 fusion activators. These cell lines exhibited extremely low basal luciferase expression but robust response to various extracellular stimuli or the expression of signaling molecules that resulted in elevated MAP kinase or PKA activities. This integrated two-component reporter system allows one to focus specifically on particular signaling pathway endpoints and the altered transactivation activity of either Elk1 or CREB. With the procedures described here, many novel cell-based assays can be developed by generating new reporter cell lines with medically important but difficult-to-transfect cell types, and by using different reporter genes or different fusion transactivator genes |
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| Beschreibung: | Date Completed 04.12.2001 Date Revised 03.12.2021 published: Print Citation Status MEDLINE |
| ISSN: | 0736-6205 |