miniCoopR Vector-based Transgenesis: A Technique to Screen Melanoma-inducing Genes in Zebrafish Tumor Model

Overview

This video describes a method to screen for the melanoma modifying genes using a transgenic zebrafish tumor model. When injected with a melanoma-inducing miniCoopR vector, the zebrafish develops melanomas and can therefore be used to study the genetic factors affecting tumor development in zebrafish.

Protocol

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

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_SV40polyA and miniCoopR to place genes of interest under the mitfa promoter in the miniCoopR vector (Figure 1A).
2. 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.
3. 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).
4. Transfer animals to 3 L tanks in the nursery of the zebrafish facility at 4 dpf.
5. 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.
6. 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.
7. 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.

Representative Results

Figure 1. Screening for melanoma onset modifiers using the miniCoopR assay. A) Schematic of the miniCoopR assay. Embryo with rescued melanocytes (arrowhead) containing the miniCoopR vector and the gene of interest. Scale bar = 250 μM. Adult with greater than 4 mm² melanocyte rescue (arrowhead). Scale bar = 500 μM B) MiniCoopR-EGFP rescued zebrafish with an amelanotic and a pigmented tumor (arrowheads). C) Representative melanoma-free survival curve comparing tumors expressing oncogene SETDB1 and a control EGFP gene (p = 9.4×10^-7, log-rank χ²).

Materials

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