靶向DNA连接酶Ⅳ小分子抑制剂SCR7提升CHO-K1细胞定点整合效率研究

Site-specific integration efficiency of CHO-K1 cells improved by DNA ligaseⅣinhibitor SCR7

目的应用DNA连接酶Ⅳ(Lig4)的小分子抑制剂SCR7处理中华仓鼠卵巢(CHO)-K1细胞,验证其能否提高CHO-K1细胞中由CRISPR/Cas9介导的外源基因在ROSA26安全位点的定点整合效率。方法构建靶向CHO-K1细胞ROSA26位点的特异性pX330-sgRNA-ROSA26质粒以及带启动子失活的GFP报告基因同源打靶载体,摸索并确定SCR7在CHO-K1细胞内作用的适宜浓度及处理时间,通过流式细胞术检测SCR7对CHO-K1细胞定点整合效率,同时评价剂量范围内SCR7对细胞凋亡的影响。结果在CHO-K1细胞中成功构建靶向ROSA26位点的GFP报告系统,该系统可用于验证CRISPR/Cas9介导的定点整合效率;确定了SCR7对CHO-K1细胞作用的合适剂量和处理时间,在该条件下SCR7对CHO-K1细胞无明显细胞毒性;证实了SCR7在剂量范围内能有效提升外源基因在CHO-K1细胞ROSA26位点的整合效率,且对细胞凋亡无明显影响。结论SCR7能提升CHO-K1细胞定点整合效率,该发现为应用Lig4小分子抑制剂构建具备高效表达外源基因的重组CHO细胞系奠定了基础。

Objective To find out whether SCR7,a small molecule inhibitor of DNA ligaseⅣ(Lig4),can improve the integration efficiency of exogenous genes at the ROSA26 safe harbor mediated by CRISPR/Cas9 in Chinese hamster ovary(CHO)-K1 cells.Methods A specific pX330-sgRNA-ROSA26 plasmid targeting the ROSA26 site in CHO-K1 cells was constructed,so was a homologous targeting vector carrying GFP gene without any promoter.Then,the appropriate conditions under which SCR7 worked in CHO cells were explored.The effect of SCR7 on site-specific integration efficiency of CHO cells was detected by flow cytometry,and the cell apoptosis induced by SCR7 was also evaluated.Results A GFP reporting system targeting ROSA26 was constructed,which could be used to analyze the efficiency of site-specific integration mediated by CRISPR/Cas9.The appropriate dose and time of SCR7 were screened out,and there was no obvious cytotoxicity on CHO-K1 cells within this dose range.It was confirmed that SCR7 could improve the integration efficiency of CHO-K1 cells at ROSA26 site.Conclusion SCR7,as a small-molecular inhibitor of Lig4,could dramatically increase the site-specific integration efficiency in CHO-K1 cells.It may be used for the construction of recombinant CHO cell lines with high expressions of exogenous genes in the future.