TOP Assay

The TOP Assay, often referred to as the TOPflash assay, is a widely used reporter gene assay in molecular biology and cell biology. It is specifically designed to measure the transcriptional activity of the Wnt/β-catenin signaling pathway. The assay utilizes a luciferase reporter construct containing multiple copies of the TCF/LEF binding site (TOP motifs) upstream of a minimal promoter driving the expression of the luciferase gene.

When the Wnt pathway is activated, β-catenin translocates to the nucleus and binds to TCF/LEF transcription factors. This complex then binds to the TOP motifs in the reporter construct, leading to increased luciferase expression. Therefore, the amount of luciferase activity detected is directly proportional to the level of Wnt/β-catenin pathway activation.

Conversely, a control reporter construct, often called FOPflash, is used to determine the basal level of transcription and the specificity of the TOPflash reporter. The FOPflash construct contains mutated TCF/LEF binding sites (FOP motifs) that prevent efficient binding of the β-catenin/TCF/LEF complex. Consequently, Wnt pathway activation does not significantly increase luciferase expression from the FOPflash reporter.

By comparing the luciferase activity from the TOPflash and FOPflash reporters, researchers can determine the fold change in Wnt/β-catenin pathway activity. This ratio helps to distinguish specific Wnt-mediated transcriptional activity from non-specific transcriptional effects.

The TOP assay is frequently employed to investigate the effects of various factors, such as Wnt ligands, receptor mutations, or pharmacological inhibitors, on Wnt pathway activity. It is a powerful tool for studying Wnt signaling in various cellular contexts and disease models, including cancer and developmental biology. It is important to note that interpretation of results must be done cautiously, considering potential off-target effects or influences from other signaling pathways.