Center for Molecular and Cellular Bioengeneering (CMCB), DFG-Center for Regenerative Therapies Dresden (CRTD), Technische Universität Dresden, Fetscherstr. 105, 01307, Dresden, Germany.
Cell Tissue Res. 2018 Apr;372(1):41-50. doi: 10.1007/s00441-018-2798-x. Epub 2018 Feb 12.
New genome-editing approaches, such as the CRISPR/Cas system, have opened up great opportunities to insert or delete genes at targeted loci and have revolutionized genetics in model organisms like the zebrafish. The Cre-loxp recombination system is widely used to activate or inactivate genes with high spatial and temporal specificity. Using a CRISPR/Cas9-mediated knock-in strategy, we inserted a zebrafish codon-optimized CreER transgene at the otx2 gene locus to generate a conditional Cre-driver line. We chose otx2 as it is a patterning gene of the anterior neural plate that is expressed during early development. By knocking in CreER upstream of the endogenous ATG of otx2, we utilized this gene's native promoter and enhancer elements to perfectly match CreER and endogenous otx2 expression patterns. Next, by combining this novel driver line with a Cre-dependent reporter line, we show that only in the presence of tamoxifen can efficient Cre-loxp-mediated recombination be achieved in the anterior neural plate-derived tissues like the telencephalon, the eye and the optic tectum. Our results imply that the otx2:CreER transgenic fish will be a valuable tool for lineage tracing and conditional mutant studies in larval and adult zebrafish.
新的基因组编辑方法，如 CRISPR/Cas 系统，为在靶基因座插入或删除基因开辟了巨大的机会，并彻底改变了斑马鱼等模式生物的遗传学。Cre-loxp 重组系统广泛用于以高时空特异性激活或失活基因。我们使用 CRISPR/Cas9 介导的基因敲入策略，将一个经过密码子优化的斑马鱼 CreER 转基因插入到 otx2 基因座，以产生条件性 Cre 驱动系。我们选择 otx2 是因为它是前神经板的模式基因，在早期发育过程中表达。通过将 CreER 敲入到 otx2 的内源性 ATG 上游，我们利用该基因的天然启动子和增强子元件，使 CreER 和内源性 otx2 的表达模式完美匹配。接下来，通过将这个新型驱动系与 Cre 依赖性报告系结合，我们表明只有在存在他莫昔芬的情况下，才能在前神经板衍生组织中实现有效的 Cre-loxp 介导的重组，如端脑、眼睛和视顶盖。我们的结果表明，otx2:CreER 转基因鱼将成为在幼鱼和成年斑马鱼中进行谱系追踪和条件性突变体研究的有用工具。
