Laboratory of Animal Disease Prevention & Control and Animal Model, The Key Laboratory of Animal Vaccine & Protein Engineering, College of Veterinary Medicine, Hunan Agricultural University (HUNAU), Changsha, Hunan, China.
Institute of Innovation and Applied Research in Chinese Medicine, Hunan University of Chinese Medicine, Changsha, Hunan, China.
PLoS One. 2020 Mar 30;15(3):e0230126. doi: 10.1371/journal.pone.0230126. eCollection 2020.
The generation of genetically modified mouse models derived from gene targeting (GT) in mouse embryonic stem (ES) cells (mESCs) has greatly advanced both basic and clinical research. Our previous finding that gene targeting at the Myh9 exon2 site in mESCs has a pronounced high homologous recombination (HR) efficiency (>90%) has facilitated the generation of a series of nonmuscle myosin II (NM II) related mouse models. Furthermore, the Myh9 gene locus has been well demonstrated to be a new safe harbor for site-specific insertion of other exogenous genes. In the current study, we intend to investigate the molecular biology underlying for this high HR efficiency from other aspects. Our results confirmed some previously characterized properties and revealed some unreported observations: 1) The comparison and analysis of the targeting events occurring at the Myh9 and several widely used loci for targeting transgenesis, including ColA1, HPRT, ROSA26, and the sequences utilized for generating these targeting constructs, indicated that a total length about 6 kb with approximate 50% GC-content of the 5' and 3' homologous arms, may facilitate a better performance in terms of GT efficiency. 2) Despite increasing the length of the homologous arms, shifting the targeting site from the Myh9 exon2, to intron2, or exon3 led to a gradually reduced GT frequency (91.7, 71.8 and 50.0%, respectively). This finding provides the first evidence that the HR frequency may also be associated with the targeting site even in the same locus. Meanwhile, the decreased trend of the GT efficiency at these targeting sites was consistent with the reduced percentage of simple sequence repeat (SSR) and short interspersed nuclear elements (SINEs) in the sequences for generating the targeting constructs, suggesting the potential effects of these DNA elements on GT efficiency; 3) Our series of targeting experiments and analyses with truncated 5' and 3' arms at the Myh9 exon2 site demonstrated that GT efficiency positively correlates with the total length of the homologous arms (R = 0.7256, p<0.01), confirmed that a 2:1 ratio of the length, a 50% GC-content and the higher amount of SINEs for the 5' and 3' arms may benefit for appreciable GT frequency. Though more investigations are required, the Myh9 gene locus appears to be an ideal location for identifying HR-related cis and trans factors, which in turn provide mechanistic insights and also facilitate the practical application of gene editing.
通过基因靶向（GT）在小鼠胚胎干细胞（mESCs）中产生的基因修饰小鼠模型的生成极大地促进了基础和临床研究。我们之前的研究发现，在 mESCs 中的 Myh9 外显子 2 位点进行基因靶向具有显著的高同源重组（HR）效率（>90%），这促进了一系列非肌肉肌球蛋白 II（NM II）相关小鼠模型的生成。此外，Myh9 基因座已被很好地证明是用于其他外源基因定点插入的新安全港。在当前的研究中，我们打算从其他方面研究这种高 HR 效率的分子生物学基础。我们的结果证实了一些以前表征的特性，并揭示了一些未报告的观察结果：1）比较和分析发生在 Myh9 基因座和几个广泛用于转基因靶向的基因座（包括 ColA1、HPRT、ROSA26 以及用于生成这些靶向构建体的序列）上的靶向事件，表明 5'和 3'同源臂的总长度约为 6kb，GC 含量约为 50%，可能有利于提高 GT 效率。2）尽管增加同源臂的长度，但将靶向位点从 Myh9 外显子 2 转移到内含子 2 或外显子 3 会导致 GT 频率逐渐降低（分别为 91.7%、71.8%和 50.0%）。这一发现首次表明，即使在同一基因座中，HR 频率也可能与靶向位点相关。同时，这些靶向位点的 GT 效率呈下降趋势与用于生成靶向构建体的序列中简单重复序列（SSR）和短散在核元件（SINE）的百分比降低一致，表明这些 DNA 元件可能对 GT 效率有潜在影响；3）我们在 Myh9 外显子 2 位点进行的一系列靶向实验和分析表明，GT 效率与同源臂的总长度呈正相关（R = 0.7256，p<0.01），证实了 5'和 3'臂的长度比为 2:1、GC 含量为 50%和 SINE 数量较高有利于可观的 GT 频率。尽管还需要更多的研究，但 Myh9 基因座似乎是鉴定 HR 相关顺式和反式因子的理想位置，这反过来为机制研究提供了见解，并促进了基因编辑的实际应用。
