Dimitrakopoulou D et al. (2019), Xenopus tropicalis: Joining the Armada in the F...

XB-ART-55712

Front Physiol 2019 Jan 01;10:48. doi: 10.3389/fphys.2019.00048.

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Xenopus tropicalis: Joining the Armada in the Fight Against Blood Cancer.

Dimitrakopoulou D , Tulkens D , Van Vlierberghe P .

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Aquatic vertebrate organisms such as zebrafish have been used for over a decade to model different types of human cancer, including hematologic malignancies. However, the introduction of gene editing techniques such as CRISPR/Cas9 and TALEN, have now opened the road for other organisms featuring large externally developing embryos that are easily accessible. Thanks to its unique diploid genome that shows a high degree of synteny to the human, combined with its relatively short live cycle, Xenopus tropicalis has now emerged as an additional powerful aquatic model for studying human disease genes. Genome editing techniques are very simple and extremely efficient, permitting the fast and cheap generation of genetic models for human disease. Mosaic disruption of tumor suppressor genes allows the generation of highly penetrant and low latency cancer models. While models for solid human tumors have been recently generated, genetic models for hematologic malignancies are currently lacking for Xenopus. Here we describe our experimental pipeline, based on mosaic genome editing by CRISPR/Cas9, to generate innovative and high-performing leukemia models in X. tropicalis. These add to the existing models in zebrafish and will extend the experimental platform available in aquatic vertebrate organisms to contribute to the field of hematologic malignancies. This will extend our knowledge in the etiology of this cancer and assist the identification of molecular targets for therapeutic intervention.

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Species referenced: Xenopus tropicalis
Genes referenced: rag2 tll1

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FIGURE 1. (A) Overview illustrating the experimental approaches to document the development of hematologic malignancies. (A) Scheme documenting the different types of analysis performed to investigate the presence of hematologic malignancies. Genotyping is performed on the blood and the dissected thymus by PCR amplification of the CRISPR/Cas9 targeted regions followed by deep sequencing of the PCR fragments and analysis of the INDEL signatures (left). Phenotyping is done by manual counting of the blood cells or by flow cytometry. In addition, lymphoid organs such as the thymus, and spleen are subjected to immunohistological analysis and transcriptomic profiling. Other organs like the kidneys and the liver are evaluated for the presence of proliferating and disseminating lymphoblasts (right). (B) Timing of the analyses that can be performed for assessing the presence of leukemic disease and to evaluate disease progression. Legend: â€œ++,â€ â€œ+â€ and â€œâ€“â€ refer to straightforward, difficult and impossible to nearly impossible to perform, respectively. Analysis can be impossible to do due to (a) immature cells jeopardizing cell discrimination, (b) too low input of thymocytes for flow cytometry in early stage tadpoles, (c) shrinking of thymi in older animals, which impedes successful dissection, (d) aberrant scattering in immature cells, (e) extremely small size of the spleen in early stage tadpoles, which therefore is difficult to dissect. Drawings adopted from Xenbase (http://www.xenbase.org/anatomy/alldev.do).

FIGURE 2. Experimental set-up for the identification of driver mutations in tumor suppressor genes (TSG) and for dependency factors. (A) Injection of Cas9 recombinant proteins and guide RNAs targeting putative TSGs created mosaic mutant animals (crispants) that contain cells with mono-allelic or bi-allelic inactivation of the driver genes. Cells with bi-allelic inactivation will have a proliferative advantages over the wild type (WT) or mono-allelic mutant cells. When disease is observed, DNA is extracted from the thymus and after PCR amplification of the targeted regions, the PCR amplicon is subjected to deep sequencing. The presence of two unique frame shifting INDELs at equal proportion is indicative of a clonally expanding subpopulation, characteristic for leukemic disease. (B) Multiplexing of guide RNAs targeting a known driver gene together with a candidate dependency factor gene. If the candidate is not a dependency factor, clonal expanding cells will be present â€“ identified by the INDEL signature of the driver gene (orange) â€“ with bi-allelic inactivation of the analyzed gene (green) (top). However, in case of a genuine dependency factor gene (purple), expanding clones will never contain bi-allelic inactivation of this gene (bottom).

References [+] :

Banach, Tumor immunology viewed from alternative animal models-the Xenopus story. 2017, Pubmed, Xenbase