慢病毒介导KIF4A稳定诱导敲降MCF7细胞株的建立  

Establishment of lentivirus-mediated KIF4A-inducible-knockdown MCF7 cell line

作　　者：闫鲁霞 程蓓蓓 周之春 朱长军[1,2,3] YAN Luxia;CHENG Beibei;ZHOU Zhichun;ZHU Changjun(College of Life Science,Tianjin Normal University,Tianjin 300387,China;Key Laboratory of Molecular and Cellular Systems Biology,Tianjin Normal University,Tianjin 300387,China;Tianjin Key Laboratory of Animal and Plant Resistance,Tianjin Normal University,Tianjin 300387,China)

机构地区：[1]天津师范大学生命科学学院,天津300387 [2]天津师范大学分子细胞系统生物学重点实验室,天津300387 [3]天津师范大学天津市动植物抗性重点实验室,天津300387

出　　处：《天津师范大学学报（自然科学版）》2022年第3期55-59,67,共6页Journal of Tianjin Normal University：Natural Science Edition

基　　金：国家自然科学基金面上资助项目(30971446);天津市科技支撑计划重点资助项目(S18ZC71124);天津市高校“十三五学科领军人才培养计划”资助项目(135205LJ24).

摘　　要：为了构建驱动蛋白KIF4A基因稳定诱导敲降细胞株Tet-shKIF4A/MCF7,应用Tet诱导基因敲降慢病毒载体系统,将慢病毒载体质粒Tet-plko-puro-shKIF4A与包装质粒psPAX2和pMD2.G共转染至293T细胞中,产生慢病毒颗粒.使用慢病毒感染MCF7细胞,用嘌呤霉素筛选出稳定诱导敲降KIF4A细胞株Tet-shKIF4A/MCF7.通过免疫印迹方法检测Tet-shKIF4A/MCF7细胞中KIF4A蛋白的表达水平,对诱导敲降KIF4A的四环素药物的浓度和敲降时间进行优化.应用免疫荧光染色的方法检测对照细胞和Tet-shKIF4A/MCF7细胞内的KIF4A的表达以及有丝分裂细胞形态的变化.结果表明,本研究成功构建了KIF4A基因稳定诱导敲降细胞株Tet-shKIF4A/MCF7,诱导敲降KIF4A的四环素药物最佳质量浓度为50μg/mL,最佳诱导时间为36 h.本研究为深入探究KIF4A在细胞生命活动中的功能以及细胞周期的分子作用机制提供了重要实验材料.To construct a stable inducible KIF4A knockdown cell line(Tet-shKIF4A/MCF7),a tetracycline-induced gene knockdown lentiviral vector system was used to transfect the lentivirus vector plasmid of Tet-plko-puro-shKIF4A into 293T cells with packaging plasmids of psPAX2 and pMD2.G to produce lentivirus particles.MCF7 cells were infected with above lentivirus and stable cell lines were screened by puromycin.Western blotting was used to examine the expression level of KIF4A protein in Tet-shKIF4A/MCF7 cells.The drug concentration and induction time of KIF4A gene knockdown were optimized.Immunofluore-scence staining was performed to detect KIF4A expression and mitotic cell morphological changes in control and Tet-shKIF4A/MCF7 cells.The results showed that the stable inducible KIF4A-knockdown cell lines(Tet-shKIF4A/MCF7)were successfully constructed.The optimal mass concentration of tetracycline and the optimal inducible time to induced KIF4A-knockdown was 50μg/mL and 36 h respectively.This work provides important experimental materials for studying the function of KIF4A in cell life as well as the molecular mechanism of cell cycle progression.

关 键 词：慢病毒 KIF4A 四环素诱导敲降系统 MCF7细胞株 

分 类 号：Q789[生物学—分子生物学] R737[医药卫生—肿瘤]