- The TOPtkLuciferase reporter assay is a specific type of luciferase reporter system designed to monitor activity of the Wnt/β-catenin signaling pathway, which plays a crucial role in cell proliferation, differentiation, and cancer development.
- The “TOP” in TOPtkLuciferase stands for “TCF Optimal Promoter”, which includes multiple TCF/LEF binding sites—the transcription factors activated downstream of Wnt signaling. These binding sites are placed upstream of a minimal thymidine kinase (tk) promoter, which drives the expression of the luciferase gene. The minimal promoter has little to no basal activity, ensuring that luciferase expression is strongly dependent on TCF/LEF-mediated transcriptional activation.
- In the assay, cells are transfected with the TOPtkLuc reporter plasmid, and then stimulated with Wnt ligands, or manipulated genetically (e.g., through β-catenin or Wnt pathway effectors like CagA from H. pylori). When Wnt signaling is active, β-catenin accumulates in the cytoplasm, translocates to the nucleus, and binds to TCF/LEF transcription factors. This complex then binds to the TCF sites in the TOPtkLuc construct, activating the tk promoter and inducing luciferase expression. The resulting luminescence, measured by a luminometer after the addition of luciferin substrate, reflects the strength of Wnt pathway activation.
- A related control reporter, FOPtkLuciferase, contains mutated TCF/LEF binding sites and serves as a negative control to confirm that any luciferase activity measured with TOPtk is specific to Wnt/TCF signaling. By comparing luciferase activity between TOPtk and FOPtk-transfected cells, researchers can quantitatively assess the activation of the canonical Wnt pathway in response to various conditions or treatments.
- The TOPtkLuciferase reporter assay is thus a valuable and widely used tool for dissecting Wnt/β-catenin signaling dynamics, particularly in studies of cancer, developmental biology, and microbial pathogenesis.
