摘要
目的:利用CRISPR/Cas9基因编辑技术构建CDH1基因缺失的人乳腺癌MCF-7稳定细胞系。方法:根据CRISPR/Cas9靶点设计原则,设计能特异性针对CDH1基因的sgRNA,以lentiCRISPR v2质粒为骨架构建能表达此sgRNA和Cas9蛋白的重组质粒。测序鉴定后,将重组质粒与逆转录病毒包装质粒VSVG、PAX2在氯化钙介导下共同转入HEK293T细胞进行病毒包装,转染48 h后收集病毒上清,直接感染人乳腺癌MCF-7细胞。采用嘌呤霉素筛选CDH1缺失的乳腺癌MCF-7细胞,通过DNA测序、Western印迹及免疫荧光染色实验验证获得的MCF-7细胞。结果:构建了靶向CDH1的CRISPR/Cas9质粒;DNA测序和Western印迹实验结果表明获得了稳定敲除CDH1的人乳腺癌MCF-7细胞。免疫荧光染色结果显示,相比对照组,稳定敲除CDH1的MCF-7细胞中已无法明显观察到E-钙黏蛋白的表达分布。结论:通过CRISPR/Cas9基因编辑技术构建了CDH1基因缺失的MCF7细胞系,为进一步研究CDH1在肿瘤免疫治疗中的作用提供了基础。
Objective:To establish a CDH1-deficient human breast cancer MCF-7 cell line by the CRISPR/Cas9 gene editing technology.Methods:A pair of sgRNAs that targeted CDH1 gene were designed and subcloned into the lentiCRISPR v2 vector to construct a recombination plasmid expressing the sgRNA and Cas9 protein according to the principles of CRISPR/Cas9 target design.After confirmed by sequencing,the recombinant plasmid together with the retroviral packaging plasmids VSVG and PAX2,were transferred into HEK293 T cells by the calcium chloride method for virus packaging.The virus supernatant was collected 48 h after transfection,and directly infected human breast cancer MCF-7 cells which thereafter were selected by puromycin.The gene knockout was examined by DNA sequencing,and the protein expression of E-cadherin in the cells was detected by Western blotting and immunofluorescence staining,respectively.Results:The CRISPR/Cas9 plasmid for CDH1 knockout was successfully constructed.DNA sequencing and Western blotting confirmed that CDH1 gene was successfully knocked out in the human breast cancer MCF-7 cells.Compared with that in the control group,the expression of E-cadherin was hardly detected in the CDH1 knockout MCF-7 cells by immunofluorescence staining.Conclusion:The MCF-7 cell clone with stable CDH1 knockout was constructed successfully by the CRISPR/Cas9 gene editing technology,which sets a basis for further research on the role of CDH1 in tumor immunotherapy.
作者
高伟健
朱一超
郑幽
张波
王征旭
孙强
GAO Wei-Jian;ZHU Yi-Chao;ZHENG You;ZHANG Bo;WANG Zheng-Xu;SUN Qiang(Medical School of Chinese PLA,Beijing 100853;Beijing Institute of Biotechnology,Beijing 100071;Air Force Medical University,Xi'an 710032;Biotherapy Center,Seventh Medical Center of PLA General Hospital,Beijing 100700,China)
出处
《生物技术通讯》
CAS
2020年第2期155-159,239,共6页
Letters in Biotechnology
