PDF(Pubmed)
Abstract：
The zebrafish is amenable to a variety of genetic approaches. However, lack of conditional deletion alleles limits stage- or cell-specific gene knockout. Here, we applied an existing protocol to establish a floxed allele for gata2a but failed to do so due to off-target integration and incomplete knockin. To address these problems, we applied simultaneous co-targeting with Cas12a to insert loxP sites in cis, together with transgenic counterscreening and comprehensive molecular analysis, to identify off-target insertions and confirm targeted knockins. We subsequently used our approach to establish endogenously floxed alleles of foxc1a, rasa1a, and ruvbl1, each in a single generation. We demonstrate the utility of these alleles by verifying Cre-dependent deletion, which yielded expected phenotypes in each case. Finally, we used the floxed gata2a allele to demonstrate an endothelial autonomous requirement in lymphatic valve development. Together, our results provide a framework for routine generation and application of endogenously floxed alleles in zebrafish.
摘要：
斑马鱼适合多种遗传方法。然而,缺乏条件缺失等位基因限制了阶段或细胞特异性基因敲除。这里,我们应用了现有的方案来建立gata2a的floxed等位基因，但由于脱靶整合和不完全敲入而未能做到这一点。为了解决这些问题,我们与Cas12a同时联合靶向在顺式中插入loxP位点，结合转基因反筛选和综合分子分析，以识别脱靶插入并确认靶向敲入蛋白。随后，我们使用我们的方法建立了foxc1a的内源性浮动等位基因，rasa1a,和ruvbl1，每一代人。我们通过验证Cre依赖性缺失来证明这些等位基因的实用性，在每种情况下都产生了预期的表型。最后,我们使用floxedgata2a等位基因来证明淋巴瓣发育过程中的内皮自主需求。一起,我们的研究结果为斑马鱼中内源性Floxed等位基因的常规生成和应用提供了框架。
