Department of Integrated Biosciences, Graduate School of Frontier Sciences, The University of Tokyo, Kashiwa, Chiba 277-8562, Japan.
RNA. 2019 Nov;25(11):1432-1438. doi: 10.1261/rna.072512.119. Epub 2019 Aug 21.
R2 is a long interspersed element (LINE) found in a specific sequence of the 28S rDNA among a wide variety of animals. Recently, we observed that R2Ol isolated from medaka fish, , retrotransposes sequence specifically into the target sequence of zebrafish. Because the 28S target and flanking regions are widely conserved among vertebrates, we examined whether R2Ol can also integrate in a sequence-specific manner in human cells. Using adenovirus-mediated expression of R2Ol constructs, we confirmed an accurate insertion of R2Ol into the 28S target of human 293T cells. However, the R2Ol mutant devoid of endonuclease (EN) activity showed no retrotransposition ability, suggesting that the sequence-specific integration of R2Ol into 28S rDNA occurs via the cleavage activity of EN. By introducing both R2Ol helper virus and donor plasmid in human cells, we succeeded in retrotransposing an exogenous EGFP gene into the 28S target site by the -complementation system, which enabled simplification of specific gene knock-in in a time-efficient manner. We believe that R2Ol may provide an alternative targeted gene knock-in method for practical applications such as gene therapy in future.
R2 是一种长散布元件（LINE），存在于多种动物的 28S rDNA 中的特定序列中。最近，我们观察到从鲤鱼中分离出的 R2Ol 能够特异性地逆转录插入斑马鱼的靶序列。由于 28S 靶序列和侧翼区在脊椎动物中广泛保守，我们研究了 R2Ol 是否也可以以序列特异性的方式整合到人细胞中。使用腺病毒介导的 R2Ol 构建体表达，我们证实 R2Ol 可以准确地插入人 293T 细胞的 28S 靶序列中。然而，缺乏内切酶（EN）活性的 R2Ol 突变体没有逆转录转座能力，这表明 R2Ol 对 28S rDNA 的序列特异性整合是通过 EN 的切割活性发生的。通过在人细胞中引入 R2Ol 辅助病毒和供体质粒，我们成功地通过 -互补系统将外源性 EGFP 基因逆转录插入 28S 靶位点，这使得通过简化特定基因敲入以更有效的方式在短时间内实现。我们相信，R2Ol 可能为未来的基因治疗等实际应用提供一种替代的靶向基因敲入方法。
