Abstract
The recent advent of CRISPR/Cas9 as a straightforward genome editing tool has allowed the establishment of the first bona fide genetic cancer models within the diploid aquatic model organism Xenopus tropicalis (X. tropicalis). Within this chapter, we demonstrate the methods for targeting tumor suppressors with the CRISPR/Cas9 system in the developing X. tropicalis embryo. We further illustrate genotyping and phenotyping of the resulting tumor-bearing F0 mosaic mutant animals (crispants). We focus in detail on the histopathological analysis of cancer neoplasms, the methodology to illustrate high proliferative index by proliferation marker immunofluorescence and how to isolate specific (tumor) cell populations by laser capture microdissection. As such, the described pipeline allows for rapid establishment of novel cancer models by CRISPR/Cas9 targeting of established tumor suppressor genes, or novel candidates obtained from clinical data. In conclusion, we thus provide the methodology for modeling human cancer with the highly efficient CRISPR/Cas9 system in F0 X. tropicalis.
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Acknowledgments
The authors would like to acknowledge Tarryn Porter (tarryn.porter@sanger.ac.uk) for allowing reuse of a figure by her hand (top part of Fig. 1). Furthermore, the authors would like to thank Dr. Tom van Nieuwenhuysen for the collaborative effort in setting up the PCNA immunofluorescence methodologies. Furthermore, the authors would like to thank Trees Lepez and prof. Dieter Deforce for the initial tutorial on how to use the PALM LCM system. Finally, the authors would like to thank Dieter Tulkens and Marjolein Carron for critical proof-reading of this chapter. Research in the authors’ laboratory is supported by the Research Foundation—Flanders (FWO-Vlaanderen) (grants G0A1515N and G029413N), by the Belgian Science Policy (Interuniversity Attraction Poles—IAP7/07) and by the Concerted Research Actions from Ghent University (BOF15/GOA/011). Further support was obtained by the Hercules Foundation, Flanders (grant AUGE/11/14), and the Desmoid Tumor Research Foundation.
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Naert, T., Vleminckx, K. (2018). Cancer Models in Xenopus tropicalis by CRISPR/Cas9 Mediated Knockout of Tumor Suppressors. In: Vleminckx, K. (eds) Xenopus. Methods in Molecular Biology, vol 1865. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-8784-9_11
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DOI: https://doi.org/10.1007/978-1-4939-8784-9_11
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