天津医药

天津医药 ›› 2016, Vol. 44 ›› Issue (10): 1190-1194.doi: 10.11958/20160731

• 细胞与分子生物学 • 上一篇    下一篇

应用CRISPR/Cas9系统在G401细胞株中敲除p21基因

赵秀娟 陈万标 张沛涛 楚晓文 张娜 楚晓文 白向阳 杨冰 吴旭东 王玺   

  1. 1 天津医科大学基础医学院细胞生物学系（邮编 300070）， 2 生物医学工程与技术学院； 3 河北省保定市莲池区凌云街 66350 部队卫生队；4 江阴迪林生物电子技术有限公司
  • 收稿日期:2016-07-26 修回日期:2016-08-24 出版日期:2016-10-15 发布日期:2016-10-21
  • 通讯作者: 王玺 E-mail:zhaoxj517@126.com
  • 作者简介:赵秀娟（1983）， 女， 博士， 讲师， 主要从事肿瘤的表观遗传学研究
  • 基金资助:
    SWI/SNF与p53相互作用对肿瘤的影响及机制研究;组蛋白去甲基化酶FBXL10在人弥漫性大B细胞淋巴瘤中的作用及机制研究

Study on p21Gene Knock Out in G401 Cell Line by Using CRISPR/Cas9 System

ZHAO Xiujuan, CHEN Wanbiao, ZHANG Peitao, ZHANG Na, CHU Xiaowen, BAI Xiangyang, YANG Bing, WU Xudong, WANG Xi   

  1. 1 Department of Cell Biology, School of Basic Medical Sciences, 2 School of Biomedical Engineering, Tianjin Medical University, Tianjin 300070, China; 3 The 66350 Military Medical Team， Lingyun Street, Lianchi District, Baoding City, Hebei Province; 4 Dealiner Bioelectronics LLC, Jiangyin
  • Received:2016-07-26 Revised:2016-08-24 Published:2016-10-15 Online:2016-10-21
  • Contact: WANG Xi E-mail:zhaoxj517@126.com

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摘要： 目的 运用 CRISPR/Cas9 基因编辑技术， 在人恶性横纹肌样瘤细胞株 G401 中敲除 p21 基因。 方法 通过反转录定量 PCR（RT-qPCR）及 Western blot 检测各瘤细胞株中 p21 的表达， 针对 p21 基因作用的功能域， 设计了靶向人 p21 基因第 3 个外显子的向导 RNA（sgRNA）， 克隆入 lentiCRISPR v2 载体。 将测序及酶切鉴定正确的重组质粒在 293T 工具细胞中制备慢病毒颗粒并感染 G401 细胞， 使用嘌呤霉素进行阳性细胞筛选， 显微镜下挑取单克隆细胞团并继续培养获得 G401 单克隆细胞株。 提取单克隆细胞株 RNA 及蛋白， 利用 RT-qPCR 及 Western blot 方法检测细胞株中 p21 的敲除效果。 结果 p21 在人横纹肌样瘤细胞中高表达。 成功构建靶向 p21 基因的 lentiCRISPR v2-sgRNA 重组慢病毒质粒。 与对照组相比， 筛选得到的 G401 亚克隆细胞系中 p21 蛋白表达缺失。 结论 针对难转染的 G401 细胞， 应用 CRISPR/Cas9 系统成功构建了 p21 基因敲除的稳定株， 为后续深入研究 p21 在人恶性横纹肌样瘤中的作用机制奠定了基础。

关键词: 横纹肌瘤, p21, 基因敲除, CRISPR/Cas9, 慢病毒

Abstract: Objective To knock out p21 gene in human malignant rhabdoid tumor（MRT） cell line G401 by using CRISPR/Cas9 genome engineering technology. Methods The expression of p21 was detected by reverse transcription quantitative PCR (RT-qPCR) and Western blot assay in several MRT cell lines. The guide RNA was designed by targeting the third exon of p21 gene， which encoded its home domains, and then subcloned into lentiCRISPR v2 vector and validated sequencing. The validated plasmids were further used to package and produce the lentivirus in 293T cells, and the G401 cells were infected, then puromycin was used to screen positive cells, and the clusters of G401 monoclonal cells, were obtained by selecting monoclonal cells and culturing under the microscope. The RNA and protein of new clonal cell line were extracted, and RT-qPCR and Western blot assay were applied to confirm whether p21 was successfully knocked out. Results The p21 was highly expressed in MRT tumor cells. The CRISPR/Cas9 lentivirus plasmids, targeted p21 gene were successfully constructed. Compared with negative control group， the expression of p21 was not detected in G401 monoclonal cells, which were successfully screened. Conclusion In view of the difficult transfection of cells such as G401, p21 knockout stable cell line has been successfully constructed by using CRISPR/Cas9 system, which lays the foundation for further study of the mechanism of p21 in MRT tumors ．

Key words: rhabdomyoma, p21, gene knockout, CRISPR/Cas9, lentivirus