Towards a zebrafish transgenic model of melanoma susceptibility

779 We are developing a model of melanoma susceptibility in zebrafish (D. rerio) in order to facilitate mapping of melanoma-susceptible and melanoma-protective traits. Evidence from murine models points to a key role in melanoma formation for inactivation of the p16INK4A/p14ARF locus combined with constitutive targeted activation of ras-mediated signalling in melanocytes. Naturally occurring, melanoma susceptible teleost hybrids (Xiphophorus spp.) confirm that similar mechanisms are likely to be operative in fish melanoma and we have begun developing transgenic zebrafish that will exhibit these features. As a first step we isolated the Nras cDNA (wt) from zebrafish total RNA and performed site-directed mutagenesis to generate the Gln61Lys (mut) mutation that is commonly observed in human tumors. Several transgenes have been constructed involving combinations of non-specific (CMV) and melanocyte-specific (nacre) promoters with either wt or mut N-ras as C-terminal fusions to YFP or GFP. Microinjected mRNA of N-ras-mut, but not N-ras–wt, resulted in early arrest of embryo development at the epiboly stage, confirming the mutant transgene is biologically active. DNA microinjections have resulted in transient expression suggestive of restriction to melanocytes and mosaic F0 adults carrying nacre-driven N-ras-mut transgenes are being screened to identify those that transmit the transgene through the germline. PCR genotyping of fin clip DNA from potential F0 founders was positive for the transgene, indicating that transgenes have incorporated into the fish genome in some potential founders. Ultimately, fish carrying the nacre-driven N-ras-mut transgenes will be exposed to UV radiation and observed for increased susceptibility to melanoma.