摘要
目的利用CRISPR/Cas9系统构建CACYBP-/-乳腺癌MCF-7基因缺陷细胞株,并研究该基因敲除株对细胞增殖能力的影响。方法设计特异sgRNA序列,构建Lenti-CAS9-sgRNA-puro质粒,转染MCF-7细胞,puromycin抗性筛选,Cruiser检测sgRNA活性,极限稀释法筛选单克隆株,Western blot检测CACYBP/SIP蛋白的表达,Brdu和MTT试剂盒检测CACYBP/SIP缺失后细胞增殖能力。结果成功构建Lenti-CAS9-sgRNA-puro质粒,puromycin最佳筛选浓度为0.9μg/mL,sgRNA1活性最高,获得的单克隆系测序唯一敲除CACYBP/SIP的MCF-7细胞株增殖能力减弱(P<0.05)。结论敲除CACYBP/SIP的MCF-7细胞株增殖能力减弱。
Objective To construct the CACYBP/SIP-/-MCF-7 cell strain and study the changes of its pro-liferation.Methods The specific sgRNA sequence was designed and Lenti-CAS9-sgRNA-puro plasmid was constructed,MCF-7 cells were transfected with the plasmid,and puromycin resistance was used to screen for positive clone.Cruiser was used to detect sgRNA activity,monoclonal strain was screened by limiting dilution method,and CACYBP/SIP protein expression was detected by Western blot,Brdu and MTT kits were used to detect cell proliferation after CACYBP/SIP gene deletion.Results A Lenti-CAS9-sgRNA-puro plasmid was successfully cons-tructed.The optimal screening concentration of puromycin was 0.9μg/mL.The results of Cruiser assay showed that sgRNA1 activity was the highest,and the obtained monoclonal strain was sequenced only.MTT and Brdu results showed that the proliferation of CACYBP/SIP-/-MCF-7 cell line was weakened(P<0.05).Conclusions CACYBP/SIP-/-MCF-7 cell strain is successfully obtained and it’s proliferation ability is decreased.
作者
王会峰
赵志军
王燕
吴媛媛
王宁菊
WANG Hui-feng;ZHAO Zhi-jun;WANG Yan;WU Yuan-yuan;WANG Ning-ju(General Hospital of Ningxia Medical UniversityDepartment of Oncology,Cancer Hospital,Yinchuan 750004,China;Department of Medical laboratory Center,Yinchuan 750004,China)
出处
《基础医学与临床》
CSCD
2019年第12期1682-1688,共7页
Basic and Clinical Medicine
基金
国家自然科学基金(81460400)
