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All procedures involving animals have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. Screening for Melanoma Onset Modifiers
- 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_SV40polyA and miniCoopR to place genes of interest under the mitfa promoter in the miniCoopR vector (Figure 1A).
- Inject 25 picograms of each clone along with 25 picograms Tol2 transposase mRNA into one-cell Tg(mitfa:BRAFV600E);p53(lf);mitfa(lf)triply homozygous zebrafish embryos as previously described. Incubate the injected embryos at 28.5 °C. Remove any dead embryos at 24 hpf.
- Select transgenic animals with rescued melanocytes at 72 hpf by placing the Petri dish containing injected embryos on a dissecting microscope under incident light against a white background (Figure 1A).
- Transfer animals to 3 L tanks in the nursery of the zebrafish facility at 4 dpf.
- At 2 months, select the animals with at least one area of melanocyte rescue greater than 4 mm2 (Figure 1A). There is a strong correlation between the degree of melanocyte rescue at 72 hpf and the degree of melanocyte rescue at 2 months. When embryos with melanocyte rescue are picked at 72 hpf, the majority of them (~80%) will have at least one area of melanocyte rescue greater than 4 mm2 at 2 months.
- Screen the selected animals weekly for the presence of tumors (Figure 1B). There is a strong correlation between histopathologic and morphologic changes. Transition from benign to malignant is recognized as a morphologic change when lesions become raised off the surface of the animal. Isolate tumor bearing animals for study.
- Draw melanoma-free survival curves with age in weeks on the abscissa and percent melanoma-free survival on the ordinate. Animals with melanocytes that express a gene of interest are compared to control animals that express EGFP (enhanced green fluorescent protein) in melanocytes (Figure 1C). The median tumor onset for animals injected with miniCoopR-EGFP is approximately 18 weeks. Determine whether the two curves are statistically different using a log rank test.
