| Autor: |
de Roo JJD; Department of Immunology, Leiden University Medical Center., Naber BAE; Department of Immunology, Leiden University Medical Center., Vloemans SA; Department of Immunology, Leiden University Medical Center., de Haas EFE; Department of Immunology, Leiden University Medical Center., van der Laan AMA; Department of Cell and Chemical Biology, Leiden University Medical Center., Staal FJT; Department of Immunology, Leiden University Medical Center; f.j.t.staal@lumc.nl. |
| Jazyk: |
angličtina |
| Zdroj: |
Journal of visualized experiments : JoVE [J Vis Exp] 2021 Sep 24 (175). Date of Electronic Publication: 2021 Sep 24. |
| DOI: |
10.3791/62141 |
| Abstrakt: |
Measuring Wnt expression levels is essential when trying to identify or test new Wnt therapeutic targets. Previous studies have shown that canonical Wnt signaling operates via a dosage-driven mechanism, motivating the need to study and measure Wnt signaling in various cell types. Although several reporter models have been proposed to represent physiological Wnt expression, either the genetic context or the reporter protein highly influenced the validity, accuracy, and flexibility of these tools. This paper describes methods for acquiring and analyzing data obtained with the Axin2-mTurquoise2 mouse Wnt reporter model, which contains a mutated Axin2 em1Fstl allele. This model facilitates the study of endogenous canonical Wnt signaling in individual cells over a wide range of Wnt activity. This protocol describes how to fully appreciate Axin2-mTurquoise2 reporter activity using cell population analysis of the hematopoietic system, combined with cell surface markers or β-catenin intracellular staining. These procedures serve as a base for implementation and reproduction in other tissues or cells of interest. By combining fluorescence-activated cell sorting and confocal imaging, distinct canonical Wnt expression levels can be visualized. The recommended measurement and analysis strategies provide quantitative data on the fluorescent expression levels for precise assessment of canonical Wnt signaling. These methods will be useful for researchers who want to use the Axin2-mTurquise2 model for canonical Wnt expression patterns. |
| Databáze: |
MEDLINE |
| Externí odkaz: |
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