Liu Tanbin, Hu Yi, Guo Shiyin, Tan Lei, Zhan Yang, Yang Lingchen, Liu Wei, Wang Naidong, Li Yalan, Zhang Yingfan, Liu Chengyu, Yang Yi, Adelstein Robert S, Wang Aibing
Lab of Animal Models and Functional Genomics (LAMFG), The Key Laboratory of Animal Vaccine & Protein Engineering, College of Veterinary Medicine, Hunan Agricultural University (HUNAU), Changsha, Hunan, China.
College of Food Science and Technology, HUNAU, Changsha, Hunan, China.
PLoS One. 2018 Feb 13;13(2):e0192641. doi: 10.1371/journal.pone.0192641. eCollection 2018.
Targeted integration of exogenous genes into so-called safe harbors/friend sites, offers the advantages of expressing normal levels of target genes and preventing potentially adverse effects on endogenous genes. However, the ideal genomic loci for this purpose remain limited. Additionally, due to the inherent and unresolved issues with the current genome editing tools, traditional embryonic stem (ES) cell-based targeted transgenesis technology is still preferred in practical applications. Here, we report that a high and repeatable homologous recombination (HR) frequency (>95%) is achieved when an approximate 6kb DNA sequence flanking the MYH9 gene exon 2 site is used to create the homology arms for the knockout/knock-in of diverse nonmuscle myosin II (NM II) isoforms in mouse ES cells. The easily obtained ES clones greatly facilitated the generation of multiple NM II genetic replacement mouse models, as characterized previously. Further investigation demonstrated that though the targeted integration site for exogenous genes is shifted to MYH9 intron 2 (about 500bp downstream exon 2), the high HR efficiency and the endogenous MYH9 gene integrity are not only preserved, but the expected expression of the inserted gene(s) is observed in a pre-designed set of experiments conducted in mouse ES cells. Importantly, we confirmed that the expression and normal function of the endogenous MYH9 gene is not affected by the insertion of the exogenous gene in these cases. Therefore, these findings suggest that like the commonly used ROSA26 site, the MYH9 gene locus may be considered a new safe harbor for high-efficiency targeted transgenesis and for biomedical applications.
将外源基因靶向整合到所谓的安全港/友好位点，具有表达正常水平的靶基因并防止对内源基因产生潜在不利影响的优点。然而，用于此目的的理想基因组位点仍然有限。此外，由于当前基因组编辑工具存在固有的且尚未解决的问题，传统的基于胚胎干细胞（ES）的靶向转基因技术在实际应用中仍然更受青睐。在此，我们报告，当使用位于MYH9基因外显子2位点侧翼的大约6kb DNA序列来创建同源臂，用于在小鼠ES细胞中敲除/敲入多种非肌肉肌球蛋白II（NM II）亚型时，可实现高且可重复的同源重组（HR）频率（>95%）。如先前所述，容易获得的ES克隆极大地促进了多种NM II基因替代小鼠模型的产生。进一步研究表明，尽管外源基因的靶向整合位点转移到了MYH9内含子2（外显子2下游约500bp处），但不仅保留了高HR效率和内源性MYH9基因的完整性，而且在对小鼠ES细胞进行的一组预先设计的实验中观察到了插入基因的预期表达。重要的是，我们证实，在这些情况下，外源基因的插入不会影响内源性MYH9基因的表达和正常功能。因此，这些发现表明，与常用的ROSA26位点一样，MYH9基因位点可被视为用于高效靶向转基因和生物医学应用的新安全港。
