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A new fluorescence-based reporter gene vector as a tool for analyzing and fishing cells with activated wnt signaling
Johanna Apfel1, Patricia Reischmann, Oliver Müller
1University of Applied Science Kaiserslautern, 66482 Zweibrücken, Germany.
Abstract:
The dysregulated Wnt pathway is a major cause for the activation of cell proliferation and reduced differentiation in tumor cells. Therefore the Wnt signaling pathway is the on-top target in searching for new anticancer drugs or therapeutic strategies. Although the key players of the pathway are known, no specific anti-Wnt drug entered a clinical trial by now. Several screening approaches for potential compounds have been performed with a reporter gene assay using multiple T-cell factor/lymphoid enhancer factor (TCF/LEF) binding motifs as promoters which control luciferase or β -galactosidase as reporter genes. In our work, we designed a reporter gene construct which anchors the enhanced green fluorescent protein (eGFP) to the plasma membrane. HEK 293T cells, which were stably transfected with this construct, express eGFP on the outer membrane after activation with either LiCl or WNT3A protein. Thus, cells with activated Wnt pathway could be identified and fished out of a heterogeneous cell pool by the use of magnetic-labeled anti-GFP antibodies. In summary, we present a new tool to easily detect, quantify, and sort cells with activated Wnt signaling pathway in a simple, fast, and cost-effective way.
Insights
Researchers developed a novel method to detect and sort cells with an activated Wnt signaling pathway. This tool uses enhanced green fluorescent protein (eGFP) on the cell surface for easy identification and isolation of Wnt-activated cells.
Area of Science:
- Molecular Biology
- Cell Biology
- Cancer Research
Background:
- Dysregulated Wnt signaling drives tumor cell proliferation and inhibits differentiation.
- The Wnt pathway is a key therapeutic target for anticancer drug development.
- Existing screening methods for Wnt pathway modulators are often indirect.
Purpose of the Study:
- To develop a novel reporter system for Wnt pathway activation.
- To enable efficient detection, quantification, and isolation of cells with activated Wnt signaling.
- To provide a cost-effective and rapid tool for Wnt pathway research.
Main Methods:
- Engineered a reporter gene construct anchoring enhanced green fluorescent protein (eGFP) to the plasma membrane.
- Stably transfected HEK 293T cells to express membrane-bound eGFP upon Wnt pathway activation.
- Utilized magnetic-labeled anti-GFP antibodies for cell sorting based on eGFP expression.
Main Results:
- Stable cell lines expressed membrane-bound eGFP when the Wnt pathway was activated by LiCl or WNT3A.
- Cells with activated Wnt signaling could be successfully identified and sorted from heterogeneous populations.
- Demonstrated a simple, fast, and cost-effective method for Wnt pathway analysis.
Conclusions:
- The developed reporter system provides a robust tool for studying Wnt pathway activation.
- This method facilitates the identification and isolation of cells with dysregulated Wnt signaling.
- Offers a valuable approach for drug screening and cancer research targeting the Wnt pathway.
