In this chapter, we are presenting methods to monitor and quantify in vivo canonical Wnt signaling activities at single-cell resolution in zebrafish. Our technology is based on artificial enhancers, obtained by polymerization of TCF binding elements, cloned upstream to ubiquitous or tissue-specific promoters. The different promoter/enhancer combinations are used to drive fluorescent protein reporter constructs integrated in the zebrafish germline by microinjection of fertilized zebrafish eggs. Fish with a single integration site are selected by Mendelian analysis of fluorescent carriers, and heterozygous offspring are used to monitor and quantify canonical Wnt activities. Open source public domain software such as ImageJ/Fiji is used to calculate the integrated densities in the region of interest and compare the effect of experimental conditions on control and treated animals.
Zebrafish Transgenic reporter Transposase Chemical biology
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The authors would like to thank Dr. Enrico Moro for his support in cloning and Dr. Tatjana Škobo for graphical help. NT is supported by the Italian Ministry of Health project Trambigen (RF-2010-2309484), the AFM-Telethon project Polygon (18572), and the UniPD grants Optozen (CPDA128151) and Tigre (CPDA148582/14). This work has been supported by CARIPARO grant 2010 “An in vivo reporter platform for cancer studies and drug screening”, AIRC Grant IG 10274 “Living reporters for glioblastoma and pancreatic adenocarcinoma” and EU grant HEALTH-F4-2010-242048 “ZF-HEALTH: Zebrafish regulomics for human health” to FA.
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