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Medicine

Screening for Melanoma Modifiers using a Zebrafish Autochthonous Tumor Model

Published: November 13th, 2012

DOI:

10.3791/50086

1Program in Molecular Medicine and Department of Cancer Biology, University of Massachusetts Medical School, 2Departments of Surgery and Medicine, Weill Cornell Medical College , 3Departments of Surgery and Medicine, New York Presbyterian Hospital

A rapid way to screen for melanoma modifiers using a zebrafish autochthonous tumor model is presented. It takes advantage of the miniCoopR vector which allows for expression of candidate melanoma genes in melanocytes. A method to obtain melanoma-free survival curves, an invasion assay, a protocol for antibody staining of scale melanocytes and a melanoma transplantation assay are described.

Genomic studies of human cancers have yielded a wealth of information about genes that are altered in tumors1,2,3. A challenge arising from these studies is that many genes are altered, and it can be difficult to distinguish genetic alterations that drove tumorigenesis from that those arose incidentally during transformation. To draw this distinction it is beneficial to have an assay that can quantitatively measure the effect of an altered gene on tumor initiation and other processes that enable tumors to persist and disseminate. Here we present a rapid means to screen large numbers of candidate melanoma modifiers in zebrafish using an autochthonous tumor model4 that encompasses steps required for melanoma initiation and maintenance. A key reagent in this assay is the miniCoopR vector, which couples a wild-type copy of the mitfa melanocyte specification factor to a Gateway recombination cassette into which candidate melanoma genes can be recombined5. The miniCoopR vector has a mitfa rescuing minigene which contains the promoter, open reading frame and 3'-untranslated region of the wild-type mitfa gene. It allows us to make constructs using full-length open reading frames of candidate melanoma modifiers. These individual clones can then be injected into single cell Tg(mitfa:BRAFV600E);p53(lf);mitfa(lf)zebrafish embryos. The miniCoopR vector gets integrated by Tol2-mediated transgenesis6 and rescues melanocytes. Because they are physically coupled to the mitfa rescuing minigene, candidate genes are expressed in rescued melanocytes, some of which will transform and develop into tumors. The effect of a candidate gene on melanoma initiation and melanoma cell properties can be measured using melanoma-free survival curves, invasion assays, antibody staining and transplantation assays.

1. Screening for Melanoma Onset Modifiers

  1. Create Gateway middle entry clones by PCR amplifying the full-length open reading frame of genes of interest (GOI) and recombining into pDONR 221 using BP clonase II (Invitrogen). Use Multisite Gateway technology (Invitrogen) to recombine p5E_mitfa, pME_GOI, Tol2kit #302 p3E_SV40polyA6 and miniCoopR5 to place genes of interest under the mitfa promoter in the miniCoopR vector (Figure 1A).
  2. Inject 25 pico.......

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One-cell Tg(mitfa:BRAFV600E);p53(lf);mitfa(lf)zebrafish embryos were injected with the miniCoopR vector containing the melanoma oncogene SETDB15 or EGFP, each under the control of the mitfa promoter. Embryos with melanocyte rescue were selected and allowed to mature. At 2 months of age animals with melanocyte rescue greater than 4 mm2 were selected. The animals were screened weekly for melanomas. Tumor incidence curves for the adults showed that the S.......

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The miniCoopR method enables expression of genes of interest in zebrafish melanocytes. This approach takes advantage of the fact that the zebrafish mitfa gene acts cell-autonomously. For this reason, melanocytes rescued by the miniCoopR vector are certain to contain the minigene and any gene of interest to which it is physically coupled. Rescued melanocytes are clearly visible and can be obtained in animals that were injected as single-cell embryos. A specified degree of chimerism is selected, and animals surpas.......

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We thank Dr. Leonard I. Zon in whose laboratory these techniques were initially developed; Kristen Kwan and the late Chi-Bin Chien for gifts of plasmids used in this work; James Lister and David Raible for assistance with antibody staining; and James Neiswender for the microinjection video. This work was funded by NIH grant R00AR056899-03 to C.J.C.

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Name Company Catalog Number Comments
Name of the reagent Company Catalogue number Comments
Gateway recombination reagents Invitrogen    
miniCoopR     Reference5
Mitfa antibody     Reference5
FITC goat anti-rabbit IgG antibody Invitrogen    
Vectashield Vector Labs H-1000  
casper Zebrafish     Reference9
701N 10 μl Syringe Hamilton/Fisher 14-824  
40 μM filter BD Falcon/Fisher 352340  
FBS Invitrogen 26140079  

  1. Beroukhim, R., et al. The landscape of somatic copy-number alteration across human cancers. Nature. 463, 899-905 (2010).
  2. Curtin, J. A., et al. Distinct sets of genetic alterations in melanoma. N. Engl. J. Med. 353, 2135-2147 (2005).
  3. Lin, W. M., et al. Modeling genomic diversity and tumor dependency in malignant melanoma. Cancer Res. 68, 664-673 (2008).
  4. Patton, E. E., et al. BRAF mutations are sufficient to promote nevi formation and cooperate with p53 in the genesis of melanoma. Curr. Biol. 15, 249-254 (2005).
  5. Ceol, C. J., et al. The histone methyltransferase SETDB1 is recurrently amplified in melanoma and accelerates its onset. Nature. 471, 513-517 (2011).
  6. Kwan, K. M., et al. The Tol2kit: a multisite gateway-based construction kit for Tol2 transposon transgenesis constructs. Dev. Dyn. 236, 3088-3099 (2007).
  7. Rosen, J. N., Sweeney, M. F., Mably, J. D. Microinjection of Zebrafish Embryos to Analyze Gene Function. J. Vis. Exp. (25), e1115 (2009).
  8. Westerfield, M. . The zebrafish book. A guide of the laboratory use of zebrafish (Danio rerio). , (2000).
  9. White, R. M., et al. Transparent adult zebrafish as a tool for in vivo transplantation analysis. Cell Stem Cell. 2, 183-189 (2008).
  10. Traver, D., et al. Transplantation and in vivo imaging of multilineage engraftment in zebrafish bloodless mutants. Nat. Immunol. 4, 1238-1246 (2003).

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