Transcription-Based Reporters of Wnt/β-Catenin Signaling
| Autor: | Allison M. Adams, Randall T. Moon, Travis L. Biechele |
|---|---|
| Rok vydání: | 2009 |
| Předmět: |
DNA
Complementary Beta-catenin Transcription Genetic Transfection General Biochemistry Genetics and Molecular Biology Cell Line chemistry.chemical_compound Genes Reporter Gene Knockdown Techniques Humans RNA Small Interfering Luciferases Gene beta Catenin Enzyme Assays biology Lentivirus RNA High-Throughput Screening Assays Cell biology Wnt Proteins Genetic Techniques chemistry Cell culture biology.protein Signal transduction DNA Signal Transduction |
| Zdroj: | Cold Spring Harbor Protocols. 2009:pdb.prot5223 |
| ISSN: | 1559-6095 |
| Popis: | INTRODUCTIONThe Wnt/β-catenin pathway is the best characterized Wnt pathway largely as a result of powerful assays that allow measurement of in vivo and in vitro pathway activation. Early assays for Wnt/β-catenin signaling included phenotypic assays in Drosophila, dorsal axis duplication in Xenopus, and proliferation of C57MG mammary epithelial cells. More recent characterizations rely on direct measurement of pathway activation. The Wnt/β-catenin signaling pathway culminates with T-cell factor/lymphoid enhancer factor (TCF/LEF)-dependent regulation of target gene transcription; assaying such transcription with reporters containing multimerized TCF/LEF DNA-binding sites is a reliable measure of pathway activation. These reporters have helped characterize the Wnt/β-catenin signaling pathway and will be critical to further studies of pathway regulation and in the development of therapeutic mechanisms to combat diseases arising from aberrant signaling. This protocol describes a variety of methods for using an improved Wnt/β-catenin reporter system, the β-catenin-activated reporter (BAR), and its accompanying control reporter system, found-unresponsive BAR (fuBAR). Procedures are presented for transient and stable transfection of the reporter. Transient transfection is very robust and does not require lentiviral production, although it has a slightly smaller dynamic range relative to stably integrated BAR. The stable transduction system can be used to generate stable reporter cell lines, assay Wnt/β-catenin signaling in cells that might be otherwise difficult to transfect, or assay signaling in vivo. The BAR system, with its improved sensitivity, greater dynamic range, and lentiviral-based reporter system, provides a vital tool for future studies of this signaling pathway. |
| Databáze: | OpenAIRE |
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