摘要
目的:应用规律成簇间隔短回文重复序列(CRISPR/Cas9)技术构建敲除BLM基因的人乳腺癌MDA-MB-231细胞株。方法:根据CRISPR/Cas9技术靶点设计原则,在BLM基因的3号外显子设计2条靶向小向导RNA(sgRNA),利用PX459 V2质粒载体,构建PX459 V2-sgRNA重组质粒;将建构成功的重组质粒转染至MDA-MB-231细胞中,通过嘌呤霉素筛选获得阳性克隆,使用Cruiser^(TM)核酸内切酶检测打靶效率,将打靶效率较高的阳性克隆进行单克隆培养,再利用免疫印迹法检测筛选出敲除BLM基因的MDA-MB-231乳腺癌细胞株。结果:测序结果表明重组质粒构建成功,免疫印迹法显示敲除BLM基因后的MDA-MB-231细胞中BLM蛋白明显降低,与空白对照组相比,BLM-KO组BLM表达水平明显低于对照组。结论:应用CRISPR/Cas9技术成功构建了BLM基因敲除的MDA-MB-231细胞株。
Objective： To knock out BLM gene in human breast cancer MDA-MB-231 cell line by using CRISPR/Cas9 genome engineering technology. Methods： According to CRISPR/Cas9 technology targets design principles,two pairs of the small guide RNA（ sgRNA） target in 3 exons of BLM were designed,and the recombinant PX459 V2-sgRNA plasmid was constructed by using PX459 V2 vectors. The MDA-MB-231 cells tranfected with the recombinant plasmid were selected by puromycin to screen the positive clone cells. The targeting efficiency was detectd by Cruiser^TM endonuclease. The high targeting efficiency positive clone was cultured,further the BLM knock-out MDA-MB-231 cell was detected by western blot. Results： Sequencing results showed that the recombinant plasmid was successful constructed. Compared with the control group,the expression of BLM in BLM-KO group was significantly lower. Conclusion： The BLM knock-out breast cancer MDA-MB-231 cell line had been successfully constructed by using CRISPR/Cas9 system,which lays the foundation for further study of the mechanism and function of BLM in breast cancer.
作者
黄晏军
郑艺
汤长宁
刘金河
刘杰麟
HUANG Yanjun;ZHENG Yi;TANG Changning;LIU Jlnhe;LIU Jlehn(Department of Immunology,Guizhou Medical University,Guiyang 550004,Guizhou,China;Tissue Eng#wering and Stem Cell Research Center,Guizhou Medical University,Guiyang 550004,Guizhou,China)
出处
《贵州医科大学学报》
CAS
2018年第5期503-507,共5页
Journal of Guizhou Medical University
基金
国家自然科学基金项目(81360349)
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黔教合人才团队字[2015]
