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Generation of clonal zebrafish lines and transplantable hepatic tumors

Nature Protocols volume 5, pages 383–394 (2010)Cite this article

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Abstract

Transplantable tumors are an accepted gold standard in cancer studies in rodents. The progress of this model in zebrafish has long been constrained by the lack of true inbred lines in zebrafish. We have generated several lines of homozygous diploid clonal zebrafish lines, which allow serial transplantations of tumor cells from one fish to another without sublethal γ-irradiation. The spectrum of transplantable tumors that were initially induced and maintained in inbred clonal zebrafish lines was limited to different types of spontaneous and diethylnitrosamine-induced hepatic tumors. However, this model can readily be extended to a broad range of extrahepatic tumors, transgenic tumors with defined mechanisms of induction and fluorescence-tagged tumor lines. These models will further facilitate in-depth analysis of invasive tumor growth, angiogenesis, metastasis and tumor-initiating cells by in vivo imaging and provide a cost-effective system for high-throughput (HTP) screening of anticancer therapeutics, including biological response modifiers. In addition, homozygous zebrafish lines are an indispensable tool for immunogenetics, mapping of quantitative trait loci and other genetic applications. The whole procedure, from generation of a gynogenetic female homozygous fish (a founder) to obtaining 3–4 consecutive passages of a syngeneic tumor, takes ∼12–18 months. This time-frame largely depends on methods of tumor induction, tumor type and tumor growth rate.

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Figure 1: Sperm collection in male zebrafish.
Figure 2: Typical abnormalities at the cleavage stage of zebrafish development.
Figure 3: Haploid syndrome.
Figure 4: Mutant (albino) and wild-type embryos 48 h post-fertilization (hpf).
Figure 5: Primary liver tumors.
Figure 6: Histological types of N-nitrosodiethylamine (DEN)-induced liver tumors.
Figure 7: Tumor transplantation, 'sheer' zebrafish.

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Acknowledgements

This work was supported in part by NCI grant CA139311 (S.R.). We thank the anonymous reviewers for their very constructive comments which provided insight and expertise that greatly assisted in making this protocol more detailed and accurate.

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Authors and Affiliations

  1. Division of Hepatology, Department of Medicine, Feinberg School of Medicine, Northwestern University, Chicago, Illinois, USA

    Igor Mizgirev & Sergei Revskoy

  2. Laboratory of Genetic Toxicology, N.N. Petrov Research Institute of Oncology, St. Petersburg, Russia

    Igor Mizgirev

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  1. Igor Mizgirev
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I.M. and S.R. contributed equally to this work.

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Correspondence to Sergei Revskoy.

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The authors declare no competing financial interests.

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Mizgirev, I., Revskoy, S. Generation of clonal zebrafish lines and transplantable hepatic tumors. Nat Protoc 5, 383–394 (2010). https://doi.org/10.1038/nprot.2010.8

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