摘要
目的:利用慢病毒介导的RNA干扰(RNAi)技术,构建稳定敲降NF-YB基因的HeLa细胞系。方法:设计shRNA-NF-YB引物,利用分子克隆技术构建H1/GFP-Puro-shRNA-NF-YB质粒,转染293T后收集病毒感染HeLa细胞;用嘌呤霉素筛选细胞得到稳定敲降细胞株;利用Western blot和实时荧光定量PCR对细胞系进行鉴定。结果:构建的质粒经测序后与设计的引物对比序列一致,Western blot和实时荧光定量PCR结果表明构建细胞的NF-YB蛋白和mRNA表达抑制效果明显。结论:本实验成功构建了NF-YB-shRNA-HeLa细胞系,获得了特异性NF-YB基因敲降的HeLa细胞。
OBJECTIVE:To construct a stably knockdown NF-YB gene in HeLa cells using the Lentivirus-mediated RNA interference(RNAi)technique.METHODS:shRNA-NF-YB primers were designed and the H1/GFP-Puro-shRNA-NF-YB plasmids were constructed by molecular cloning.The virus-infected HeLa cells were collected after transfection with 293T.Stably knock down cells were obtained by screening cells with puromycin.Cell lines were identified by Western blot and qPCR.RESULTS:The constructed plasmid was sequenced by the company and was consistent with the designed primer sequence.Western blot and qPCR results show that inhibition of NF-YB protein and mRNA expression was significant.CONCLUSION:In this experiment,NF-YB-shRNA-HeLa cell line was successfully constructed and HeLa cells with specific NF-YB gene silencing knockdown were obtained.
作者
杜璐
姜晓燕
丁库克
DU Lu;JIANG Xiaoyan;DING Kuke(National Institute for Radiological Protection,Chinese Center for Disease Control and Prevention,Beijing 100088,China)
出处
《癌变．畸变．突变》
CAS
2020年第6期409-413,共5页
Carcinogenesis,Teratogenesis & Mutagenesis
基金
国家自然科学基金(31770907)
北京市自然科学基金(7172146)。
