Development of genome-modified Xiphophorus

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Project Summary: Xiphophorus fishes are best known as long-standing biomedical models for translational research on cancer development, photobiology and metabolism. They are widely used in research topics that connect to the missions of multiple NIH Institutions/Centers. Xiphophorus have been used for behavioral (NIMH), evolutionary (NHGRI), physiological (NIGMS), developmental, toxicological (NIEHS), genomics (NHGRI), immunological (NIAID), and parasitological studies (NIAID), dietary effects on metabolism (NIDDK), sex determination and gonad development (NICHD), maturation (NICHD), chronobiology (NIGMS), cancer etiology (NCI), and optical genetic regulation (NIGMS). Xiphophorus fish exhibit several features that no other animal models share, including a high level of inter-species genetic divergence, the capability of producing inter-species and backcross hybrids, and adaptive phenotypes representing several human diseases. Xiphophorus is an evolutionary mutant model that is particularly suited to search for disease-modifying factors. Recent progress in high-throughput genomic methods has accelerated the detection of candidate disease-causing genes involved e.g. in modifying tumor progression, in obesity, heart diseases or immunity regulation. These and more genes await functional validation and molecular mechanism characterization to promote them to translational studies. Because Xiphophorus is a live-bearing fish species, transgenic technologies that have been developed in egg-laying model species cannot be utilized for Xiphophorus because such methods work only in the one-cell stage embryo, which cannot be isolated and cultured in Xiphophorus. Therefore, there is an urgent necessity to develop an alternative technique of modifying the genome of live-bearing fish for versatile applications. We propose as an innovative approach to use lentiviral transduction for gene transfer to late-stage Xiphophorus embryos, which can be cultured in vitro. To accomplish this, we will improve the in vitro culture conditions to a point where a sufficiently high number of genome- modified embryos will develop to fertile adults and give rise to stable genetically altered lines. The deliverable is a protocol that can be readily adopted by other laboratories and can be applied to other livebearing fish models. For the proof-of-concept, we will use the CRISPR/Cas-9 system to knock-out a Xiphophorus pigmentation-driving and cancer-predisposing gene, xmrk, that will result in an engineered fish that is resistant to crossing-conditioned melanoma. We will deliver a new Xiphophorus line that will be useful for studies on skin cancer and a protocol for genome modification of live-bearing fish. While Xiphophorus has since decades served mainly as a model in cancer research, and more recently also for obesity, the establishment of transgenic technologies will open the next chapter of research with this model and boost its usage for additional biomedical research through the possibility to generate new disease models by genome modification. The outcome of the proposal will enhance the use of the Xiphophorus model and ultimately lead to a better understanding of human disease with impacts on diagnosis and therapeutic strategies.