稳定敲降NF-YB基因HeLa细胞系的构建

doi:10.3969/j.issn.1004-616x.2020.06.001

癌变·畸变·突变 ›› 2020, Vol. 32 ›› Issue (6): 409-413.doi: 10.3969/j.issn.1004-616x.2020.06.001

• 论著 • 下一篇

稳定敲降NF-YB基因HeLa细胞系的构建

杜璐, 姜晓燕, 丁库克

中国疾病预防控制中心辐射防护与核安全医学所放射生态研究室, 北京 100088

收稿日期:2020-04-14 修回日期:2020-09-01 出版日期:2020-12-01 发布日期:2020-12-04
通讯作者: 丁库克,E-mail:shouding@ccmu.edu.cn E-mail:shouding@ccmu.edu.cn
作者简介:杜璐,E-mail:1136856133@qq.com。
基金资助:
国家自然科学基金（31770907）；北京市自然科学基金（7172146）

Stable knockdown of NF-YB gene in the HeLa cell line

DU Lu, JIANG Xiaoyan, DING Kuke

National Institute for Radiological Protection, Chinese Center for Disease Control and Prevention, Beijing 100088, China

Received:2020-04-14 Revised:2020-09-01 Online:2020-12-01 Published:2020-12-04

摘要/Abstract

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。

关键词: NF-YB基因, RNA干扰, HeLa细胞, 慢病毒介导

Abstract: 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.

Key words: NF-YB gene, RNA interference, HeLa cells, lentivirus-mediated

中图分类号:

Q813

杜璐, 姜晓燕, 丁库克. 稳定敲降NF-YB基因HeLa细胞系的构建[J]. 癌变·畸变·突变, 2020, 32(6): 409-413.

DU Lu, JIANG Xiaoyan, DING Kuke. Stable knockdown of NF-YB gene in the HeLa cell line[J]. Carcinogenesis, Teratogenesis & Mutagenesis, 2020, 32(6): 409-413.

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稳定敲降NF-YB基因HeLa细胞系的构建

Stable knockdown of NF-YB gene in the HeLa cell line

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