利用CRISPR/Cas9系统构建4T1细胞CXCR4基因敲除稳定细胞株

目的 利用CRISPR/Cas9系统敲除4T1细胞中的CXCR4基因，构建稳定敲除CXCR4基因的4T1细胞株。 方法 根据CRISPR/Cas9靶点设计原则，在美国国立生物技术信息中心（NCBI）上找到CXCR4基因序列的外显子区域，设计两条sgRNA，用LentiCRISPRv2作为载体构建LentiCRISPRv2-sgRNA重组质粒并转化至感受态的Stbl3菌体中，挑取单克隆测序验证并扩大培养提质粒后转染至293T细胞中包装成慢病毒。 收集病毒并感染4T1细胞，通过嘌呤霉素筛选并用有限稀释法分离培养出单克隆细胞。 提取筛选出的单克隆细胞基因组DNA并对敲除位点附近的DNA片段进行PCR扩增并测序；用Real-time PCR检测细胞株CXCR4基因mRNA表达情况；用免疫印迹法检测CXCR4蛋白质的表达情况。 结果 LentiCRISPRv2-sgRNA重组质粒构建成功；经过基因组DNA片段PCR扩增测序得1株缺失27 bp的稳定敲除CXCR4基因的细胞株；细胞株CXCR4mRNA的表达量低且几乎无CXCR4蛋白质的表达。 结论 通过CRISPR/Cas9系统获得靶向敲除CXCR4基因的重组质粒，并筛选出稳定敲除CXCR4基因的细胞株。

Construction of 4T1 CXCR4 knockout gene stable strain using CRISPR/Cas9 gene editing system

Objective To knock out CXCR4 gene in 4T1 cell by CRISPR/Cas9 system and to construct 4T1 cell strain with knocking out CXCR4 gene stably. Methods Two pairs of sgRNAs that could specifically identify the exons of CXCR4 gene were designed to construct LentiCRISPRv2-sgRNA recombinant plasmid and transformed into competent Stbl3.Then the recombinant plasmids were screened for sequencing and transfected into 293T cell to packaging into lentivirus. After infection of 4T1 cells with lentivirus, stable transfected cells were selected by puromycin,and monoclonal cells were isolated and cultured by limiting dilution method . The genomic DNA of monoclonal cells was extracted and sequenced. The expression of CXCR4 mRNA was detected by Real-time PCR. The expression of CXCR4 protein was detected by Western blotting. Results The LentiCRISPRv2-sgRNA recombinant plasmid was successfully constructed, and a cell strain of knocking out CXCR4 deletion 27 bp was obtained. The expression level of CXCR4 mRNA was reduced, and there was almost no expression of CXCR4 protein in cell strain. Conclusion Recombinant plasmids targeting the CXCR4 gene was obtained by CRISPR/Cas9 gene edit system, and the cell strain with stable knockout CXCR4 gene was successfully screened out.