Although zebrafish are well suited for transgenic manipulation, routine transgenesis in zebrafish with random DNA integration presents considerable challenges. A study in Science Advances reports the development of a new transgenesis approach using well-validated ‘safe harbor’ sites in the zebrafish genome for reproducible, routine incorporation of transgenes into predictable loci. Lalonde et al. adapted a method previously described in mouse and Drosophila transgenesis, in which phiC31 integrase – a recombinase from the bacteriophage phiC31 – is expressed to mediate the integration of a donor plasmid containing an attB sequence into a genomic attP landing site. Here, the investigators used a CRISPR/Cas9 approach to create attP landing sites into the zebrafish genome and showed that the phiC31 Integrase Genomic Loci Engineered for Transgenesis (pIGLET) landing sites facilitated diverse transgenesis applications, including integration of reporters and Cre/loxP transgenes. The method, which consistently yielded 25 to 50% germline transmission from F0 zebrafish, could substantially reduce the workload and resources needed for zebrafish transgenic line generation.

Original reference: Lalonde, L.R. et al. Sci. Adv. 10, eadn6603 (2024)

尽管斑马鱼非常适合转基因操作，但随机 DNA 整合斑马鱼的常规转基因提出了相当大的挑战。 《科学进展》中的一项研究报告了一种新的转基因方法的开发，该方法利用斑马鱼基因组中经过充分验证的“安全港”位点，将转基因可重复地、常规地掺入可预测的基因座中。拉隆德等人。采用了先前在小鼠和果蝇转基因中描述的方法，其中表达 phiC31 整合酶（一种来自噬菌体 phiC31 的重组酶）以介导包含attB序列的供体质粒整合到基因组attP着陆位点。在这里，研究人员使用 CRISPR/Cas9 方法在斑马鱼基因组中创建att P着陆位点，并表明 phiC31 整合酶基因组基因座转基因工程 (pIGLET) 着陆位点促进了多种转基因应用，包括报告基因和 Cre/loxP 转基因的整合。该方法始终能从 F0 斑马鱼获得 25% 至 50% 的种系传播，可以大大减少斑马鱼转基因系生成所需的工作量和资源。

原始参考文献： Lalonde, LR et al 。科学。 10 、eadn6603（2024）