应用规律成簇的间隔短回文重复/Cas9构建敲除趋化因子受体4的人胶质瘤U251细胞

Construction of chemokine receptor 4 knockout glioma cell line U251 by clustered regularly interspaced short palindromic repeats/Cas9

目的 运用规律成簇的间隔短回文重复（CRISPR）/Cas9基因编辑技术构建敲除趋化因子受体4 （CXCR4）基因的人脑胶质瘤U251细胞.方法 设计3对针对目标基因CXCR4的导向RNA（sgRNA）,通过PX335质粒构建3组该基因的Cas9表达载体（CXCR4-sgRNA-1、CXCR4-sgRNA-2、CXCR4-sgRNA-3）,测序验证后,将构建正确的载体CXCR4-sgRNA-1转染进至U251细胞,T7核酸内切酶Ⅰ （T7E1）酶切鉴定敲除效率,分离单克隆细胞5组进行培养,分别测序及蛋白印迹法鉴定其敲除效率和蛋白含量,获得稳定敲除阳性单克隆细胞.结果 测序验证CXCR4-sgRNA-1组载体构建成功,转染载体后U251细胞经聚合酶联反应（PCR）酶切鉴定敲除效率为48％,其2、3、4、5号单克隆细胞敲除效率分别为20％、40％、30％、20％.PCR产物连接T载体后测序,结果显示3组出现点突变,2号为A突变为G,4号为T突变为C和5号为A突变为G,而3号单克隆敲入38 bp碱基,确定为有意义的突变.2、3、4号单克隆细胞CXCR4蛋白表达量为0,敲除组CXCR4蛋白相对表达量较对照组明显降低（0比0.746±0.010,P=0.000）,差异有统计学意义.免疫印迹结果说明敲除CXCR4基因的U251细胞株内无CXCR4蛋白的表达;测序比对得到有效的敲除细胞株.结论 通过CRISPR/Cas9系统高效地获得了靶向CXCR4重组质粒,并且筛选出稳定敲除CXCR4表达的U251细胞株.

Objective To construct human glioma U251 cells with knockout of the chemokine receptor 4 （CXCR4） gene using clustered regularly interspaced short palindromic repeats/Cas9 （CRISPR/Cas9） gene editing techniques.Methods Three pairs of directed RNA [single guide RNA （sgRNA）] for target gene CXCR4 have been designed,then three groups of Cas9 expression vectors （CXCR4-sgRNA-1,CXCR4-sgRNA-2,CXCR4-sgRNA-3） were constructed by PX335 plasmid.They were sequenced and verified.The right carrier （CXCR4-sgRNA-1） was chosen and transfected into U251 cells.T7 endonuclease Ⅰ （T7E1） enzyme digestion was used to determine the knockout efficiency,and 5 groups of monoclonal cells were obtained.By sequencing and Western blotting,we obtained stable positive monoclonal cells.Results The CXCR4-sgRNA-1 group was successfully constructed.PCR enzyme digestion determined that the knockout efficiency of U251 cells after transfection was 48%,and that of no.2,3,4 and 5 monoclonal cells was 20%,40%,30% and 20% respectively.The PCR product was sequenced after connecting the T vector,and the results showed that point mutation occurred in three groups.No.2 had a mutation from A to G,no.4 T to C and No.5 A to G.A 38 bp base was inserted into the no.3 McAb,and a meaningful mutation was identified.The CXCR4 protein expression in no.2,3,and 4 monoclonal cells was 0.The results of immunoblotting showed that the expression of CXCR4 protein was not detectable in the U251 cells with knockout of the CXCR4 gene,the relative expression of CXCR4 protein in knockout group was significantly lower than that of the control group （0 to 0.746 ± 0.010,P =0.000）,the difference was statistically significant.An effective knockout cell line was obtained by sequencing comparison.Conclusion The recombinant plasmid targeting CXCR4 was obtained efficiently through CRISPR/Cas9 system,and a stable U251 cell line with knockdown of CXCR4 gene was screened out.