HMEJ-mediated efficient site-specific gene integration in chicken cells
HMEJ-mediated efficient site-specific gene integration in chicken cells
复制标题
HMEJ 介导的鸡细胞中有效的位点特异性基因整合。
DOI：
10.1186/s13036-019-0217-9
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发表时间：
2019-11-21
影响因子：
5.6
通讯作者：
Lu, Yangqing
中科院分区：
文献类型：
--
作者：
Xie, Long;Sun, Juanjuan;Lu, Yangqing
转基因鸡细胞的生产在包括发育生物学和生物医学研究在内的几个不同领域具有很大的前景。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，并已成功地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。本研究比较了基于同源非依赖性靶向整合（HITI）、同源定向修复（HDR）和同源介导末端连接（HMEJ）的基因敲入方法在鸡DF-1细胞和原始生殖细胞（PGCs）中的应用，并结合集群规则间隔短回复性重复相关蛋白9 （CRISPR/Cas9）基因编辑系统。HMEJ是鸡PGCs基因敲入的一种稳健有效的方法。利用这种方法，我们成功地标记了鸡PGCs中的生殖细胞特异性基因DAZL和多能性相关基因Pou5f3，通过在基因3 '端的框架中插入荧光蛋白，使我们能够跟踪细胞在胚胎性腺中的迁移。HMEJ策略也成功地应用于蛋清蛋白（Ovalbumin）中，蛋清蛋白占鸡蛋蛋白质的60%以上，这表明它在利用鸡输卵管系统大规模生产具有药用价值的蛋白质方面具有良好的前景。综上所述，HMEJ可有效介导鸡PGCs的位点特异性基因整合，在鸡细胞的生物制药工程中具有很大的应用潜力。
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.