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

- In zebrafish, melanoma arises from the malignant transformation of melanocytes - the melanin-producing cells. To study melanoma onset, use a melanoma-prone, melanocyte deficient transgenic zebrafish model. This model lacks melanocytes due to a loss-of-function mutation in its melanocyte-specific mitfa promoter.

Begin by taking a single-celled embryo of this transgenic model on an agar plate. Co-inject a mix containing a transposon-based miniCoopR vector and transposase enzyme mRNAs into the embryo. The vector contains a gene cassette, comprising a promoter-carrying mitfa minigene coupled to a candidate gene sandwiched between two transposon elements.

The co-injected mRNAs encode transposase proteins. These proteins act on the transposon elements and catalyze the cassette excision from the miniCoopR vector, followed by its integration into the genomic DNA of the embryo. Subsequently, the cassette promoter drives the expression of both the mitfa gene and the candidate gene of interest.

The mitfa gene supports melanocyte rescue and development, while the candidate gene, if oncogenic, induces melanoma onset. Screen the developed zebrafish. Transgenic fish with rescued melanocytes develops melanin pigmentation, while those bearing melanomas carry raised pigmented lesions.

In the following protocol, we will show the screening of melanoma onset modifiers using miniCoopR vectors in transgenic zebrafish tumor models.

- To generate constructs for injecting, begin by creating Gateway middle entry clones by PCR amplifying the full-length open reading frame of genes of interest and recombining into pDONR221. Then, use Multisite Gateway technology to recombine the melanocyte-specific mitfa promoter, gene of interest, and polyadenylation signal into the miniCoopR vector to place genes of interest under the control of the mitfa promoter.

Inject 25 picograms of each clone along with 25 picograms of Tol2 transposase mRNA into one-cell, transgenic, triple-homozygous zebrafish embryos. mitfa-BRAF-p53 mutant animals are melanocyte deficient and prone to transformation and melanoma formation. Along with the gene of interest, the miniCoopR vector contains a rescuing mitfa minigene. So, successfully injected animals will develop rescued melanocytes, which express the gene of interest.

Incubate the injected embryos at 28.5 degrees Celsius. At 24 hours post-fertilization, or hpf, remove any dead embryos. At 72 hours post-fertilization, using a dissecting microscope under incident light against a white background, select transgenic animals with rescued melanocytes.

When the animals reach 4 days post-fertilization, transfer them to 3-liter tanks in the nursery of the zebrafish facility. At 2 months of age, select the animals with at least one area of melanocyte rescue greater than 4 millimeters squared. Screen the selected animals weekly for the presence of tumors. Isolate tumor-bearing animals for study.

Draw melanoma-free survival curves with age and weeks on the abscissa and percent melanoma-free survival on the ordinate. Compare animals with melanocytes that express a gene of interest to control animals that express EGFP in melanocytes. Use a log-rank test to determine whether the two curves are statistically different.