Versatile <i>Xenopus tropicalis</i> model with targeted integration of human <i> BRAF ^V600E </i>

Targeting exogenous gene integrations in animals often exhibits low efficiency, limiting the development of gene knock-in models. Theoretically, by screening founder generation individuals based on the cell phenotypes resulting from gene knock-ins and leveraging the high fecundity of animals, heritable descendants with targeted knock-ins can be efficiently generated. Therefore, we utilized the high fecundity of <i>Xenopus tropicalis</i> and easily observable pigment phenotypes to construct a <i>BRAF^V600E</i>-targeted <i>mitf</i> locus knock-in model. Results indicated that this approach enabled efficient generation of <i>BRAF^V600E</i> knock-in <i>X. tropicalis</i> and produced a versatile frog model. The <i>BRAF^V600E</i> knock-in induced the transdifferentiation of RPE cells into retinal cells, resulting in a symmetric retinal structure in the eyes of these frogs. The transformation of RPE cells ultimately leads to these frogs becoming eyeless frogs, which serve as a tool for retinal regeneration research. Additionally, in eyeless frogs the <i>BRAF^V600E</i> knock-in led to the abnormal proliferation of both melanocytes and xanthophores into melanocytic and xanthocytic nevi respectively. Consequently, eyeless frogs provide a model for studying abnormal pigment cell proliferation, offering a platform for investigating pigment cell nevus formation. Furthermore, the <i>cdkn2b</i>-knockout eyeless frogs serve as a valuable xanthophoroma model for tumor biology research. Overall, the <i>BRAF^V600E</i>-targeted knock-in <i>X. tropicalis</i> not only represents a strategy for constructing gene knock-in animal models but also serves as a versatile tool for research in retinal regeneration and tumor biology.