稳定干扰核糖体蛋白RPS7基因表达的宫颈癌HeLa细胞株的建立 |
| |
引用本文: | 丁慧,禹立霞,凌静娴,刘宝瑞,张葵,王庆飞.稳定干扰核糖体蛋白RPS7基因表达的宫颈癌HeLa细胞株的建立[J].国外医学:分子生物学分册,2013(6):318-322. |
| |
作者姓名: | 丁慧 禹立霞 凌静娴 刘宝瑞 张葵 王庆飞 |
| |
作者单位: | [1]南京大学医学院附属鼓楼医院肿瘤科,南京市210008 [2]南京大学医学院附属鼓楼医院妇科,南京市210008 [3]南京大学医学院附属鼓楼医院检验科,南京市210008 |
| |
基金项目: | 国家自然科学基金(No.81302241),南京市医学科技发展专项 |
| |
摘 要: | 目的建立稳定抑制RPS7基因表达的宫颈癌HeLa细胞株。方法设计并合成靶向人RPS7基因的shRNA寡核苷酸片段,克隆到逆转录病毒载体pSIREN中,构建重组质粒pSIREN-RPS7-shRNA,转染293T细胞,将包装产生的重组逆转录病毒感染宫颈癌HeLa细胞,经嘌呤霉素筛选获得稳定的细胞克隆,用real-timePCR和Western印迹检测细胞中RPS7mRNA和蛋白表达水平。结果获得了经测序鉴定正确的重组逆转录病毒质粒,逆转录病毒感染HeLa细胞后用嘌呤霉素筛选出的稳定细胞中,RPS7mRNA和蛋白水平均显著低于干扰对照细胞。结论成功构建了靶向人RPS7基因的shRNA逆转录病毒载体,建立了稳定抑制RPS7基因表达的宫颈癌HeLa细胞株.为进一步研究RPS7在宫颈癌中的生物学功能和作用机制提供了可靠的细胞模型。
|
关 键 词: | RPS7基因 shRNA 逆转录病毒载体 宫颈癌 |
Establishment of HeLa Cervical Cancer Cell Linewith Stable Knock- down of RPS7 |
| |
Authors: | DING Hui YU Lixia LING Jingxian LIU Baorui ZHANG Kui WANG Qingfei |
| |
Institution: | 1The Comprehensive Cancer Center, 2Department of 3 Gynecology, Department of Laboratory Medicine, Nanjing Drum Tower Hospital, School of Medicine, Nanjing University, Nanjing, 210008, China) |
| |
Abstract: | Objective human ribosomal protein S7 To establish a HeLa cervical cancer cell line with stable knockdown of gene (RPS7) . Methods shRNA sequences targeting RPS7 were de- signed and cloned into the retrovirus vector pSIREN to obtain the recombinant plasmids pSIREN- RPS7-shRNA. The recombinant plasmids were thereafter transfected into 293T cells for virus pack- age, which was followed by infection of HeLa cells with the packaged virus. Then puromycin was used to generate the stable virus-infected HeLa cells. The RPS7 gene expression level was measured by real-time PCR and Western blotting. Results Recombinant retrovirus shRNA plasmids were cor- rectly constructed as confirmed by sequencing results. RPS7 mRNA and protein levels were signifi- cantly decreased in puromycin-treated pSIREN-RPS7-shRNA- transfected HeLa cells compared with control cells. Conclusions The recombinant retrovirus shRNA vectors targeting RPS7 were success- fully constructed and HeLa cell models with stable knockdown of RPS7 were established, which pro- vide a useful cell line model to further investigate the biological roles of RPS7 and its action mecha- nisms in cervical cancers. |
| |
Keywords: | RPS7 gene shRNA retrovirus cervical cancer |
本文献已被 维普 等数据库收录! |