Background The production of transgenic chicken cells holds great promise for several diverse areas, including developmental biology and biomedical research. To this end, site-specific gene integration has been an attractive strategy for generating transgenic chicken cell lines and has been successfully adopted for inserting desired genes and regulating specific gene expression patterns. However, optimization of this method is essential for improving the efficiency of genome modification in this species. Results Here we compare gene knock-in methods based on homology-independent targeted integration (HITI), homology-directed repair (HDR) and homology mediated end joining (HMEJ) coupled with a clustered regularly interspaced short palindromic repeat associated protein 9 (CRISPR/Cas9) gene editing system in chicken DF-1 cells and primordial germ cells (PGCs). HMEJ was found to be a robust and efficient method for gene knock-in in chicken PGCs. Using this method, we successfully labeled the germ cell specific gene DAZL and the pluripotency-related gene Pou5f3 in chicken PGCs through the insertion of a fluorescent protein in the frame at the 3' end of the gene, allowing us to track cell migration in the embryonic gonad. HMEJ strategy was also successfully used in Ovalbumin, which accounts for more than 60% of proteins in chicken eggs, suggested its good promise for the mass production of protein with pharmaceutical importance using the chicken oviduct system. Conclusions Taken together, these results demonstrate that HMEJ efficiently mediates site-specific gene integration in chicken PGCs, which holds great potential for the biopharmaceutical engineering of chicken cells.

中文翻译：

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HMEJ 介导的鸡细胞中有效的位点特异性基因整合。

背景 转基因鸡细胞的生产在发育生物学和生物医学研究等多个领域具有广阔的前景。为此，位点特异性基因整合已成为产生转基因鸡细胞系的一种有吸引力的策略，并已成功用于插入所需基因和调节特定基因表达模式。然而，该方法的优化对于提高该物种基因组修饰的效率至关重要。结果在这里，我们比较了基于同源独立靶向整合（HITI）、同源定向修复（HDR）和同源介导末端连接（HMEJ）以及簇状规则间隔短回文重复相关蛋白9（CRISPR/ Cas9) 鸡 DF-1 细胞和原始生殖细胞 (PGC) 中的基因编辑系统。HMEJ 被发现是一种稳健且有效的鸡 PGC 基因敲入方法。利用该方法，我们通过在鸡 PGC 的 3' 端框架中插入荧光蛋白，成功标记了鸡 PGC 中的生殖细胞特异性基因 DAZL 和多能性相关基因 Pou5f3，从而使我们能够追踪鸡 PGC 中的细胞迁移。胚胎性腺。HMEJ 策略也成功应用于卵清蛋白（鸡蛋中蛋白质含量超过 60%），这表明该策略对于利用鸡输卵管系统大规模生产具有药用价值的蛋白质具有良好的前景。结论 综上所述，这些结果表明 HMEJ 有效介导鸡 PGC 中的位点特异性基因整合，这对于鸡细胞的生物制药工程具有巨大的潜力。