Versatile knock-in Xenopus tropicalis

What is it about?

The targeted integration of exogenous genes in animals is generally inefficient, which limits the development of gene knock-in animal models. In theory, by screening founder individuals based on gene knock-in–induced cellular phenotypes and leveraging the high reproductive capacity of animals, it is possible to efficiently obtain heritable offspring carrying precise knock-ins. On this basis, this study took advantage of the high fecundity of Xenopus tropicalis and its easily observable pigmentation phenotypes to efficiently generate a BRAFV600E knock-in model targeted to the mitf locus. In summary, the efficiently established BRAFV600E knock-in Xenopus tropicalis not only provides a strategy for developing gene knock-in animal models but also serves as a versatile tool for research on retinal regeneration and tumor biology.

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Photo by Sangharsh Lohakare on Unsplash

Photo by Sangharsh Lohakare on Unsplash

Why is it important?

We established a novel approach for generating human disease animal models by targeting the human BRAFV600E mutation into the mitf locus of Xenopus tropicalis, thereby creating a versatile and heritable disease model. On this knock-in basis, we simultaneously obtained a Xenopus melanoma model and an eyeless model for retinal regeneration. This study uncovered several biological and methodological insights. First, we report for the first time a vertebrate BRAFV600E knock-in–induced retinal regeneration model, demonstrating that activation of the BRAF/MAPK signaling pathway can drive retinal pigment epithelial cells to transdifferentiate into retinal cells, and further revealing its role in cell-fate conversion between melanophores and xanthophores. Second, we observed distinct immune infiltration patterns: CD3-positive T cells were enriched in nevi, whereas xanthophoromas exhibited immune exclusion. Third, we developed a cost-effective knock-in strategy based on phenotypic screening, taking full advantage of the high fecundity of Xenopus. Fourth, functional analysis of the mitf-BRAFV600E+/mitf- line revealed that C-terminal peptide fusion compromises Mitf protein function, providing critical guidance for the design of future knock-in constructs. Together, these findings highlight the complexity of endogenous transcriptional regulation, underscoring that transgene expression driven solely by exogenous promoters cannot fully recapitulate endogenous gene expression patterns in vivo. Moreover, our results offer important theoretical and technical references for research in retinal regeneration, pigment cell biology, cell-fate determination, tumor biology, and the development of vertebrate disease models.

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