Details

Autor(en) / Beteiligte

• Biechele, Travis L
• Adams, Allison M
• Moon, Randall T

Titel

Transcription-based reporters of Wnt/beta-catenin signaling

Ist Teil von

• Cold Spring Harbor protocols, 2009-06, Vol.2009 (6), p.pdb.prot5223

Ort / Verlag

United States

Erscheinungsjahr

2009

Quelle

MEDLINE

Beschreibungen/Notizen

• INTRODUCTION The 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.