[1]王　晶,高　倩,陈健康,等.应用CRISPR/Cas9基因编辑系统对HEK293细胞系TSC1基因稳定敲除效果的实验鉴定[J].现代检验医学杂志,2021,36(04):129-131.[doi:10.3969/j.issn.1671-7414.2021.04.027]
　WANG Jing,GAO Qian,CHEN Jian-kang,et al.Construction and Identification of TSC1 Gene Knockout Stable Cell Line inHEK293 Using CRISPR/Cas9 Gene Editing System[J].Journal of Modern Laboratory Medicine,2021,36(04):129-131.[doi:10.3969/j.issn.1671-7414.2021.04.027]
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应用CRISPR/Cas9基因编辑系统对HEK293细胞系TSC1基因稳定敲除效果的实验鉴定()
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《现代检验医学杂志》[ISSN:/CN:]

卷:
第36卷
期数:
2021年04期
页码:
129-131
栏目:
研究简报·实验技术
出版日期:
2021-07-31

文章信息/Info

Title:
Construction and Identification of TSC1 Gene Knockout Stable Cell Line inHEK293 Using CRISPR/Cas9 Gene Editing System
文章编号:
1671-7414（2021）04-129-04
作者:
王　晶; 高　倩; 陈健康; 刘家云
（空军军医大学第一附属医院临床检验研究所，西安 710032）
Author(s):
WANG Jing;  GAO Qian;  CHEN Jian-kang;  LIU Jia-yun
(Research Institute of Clinical Laboratory Medicine, the First Affiliated Hospital of the Air Force Medical University,Xi’an 710032, China)
关键词:
成簇规律间隔短回文重复序列(CRISPR)/CRISPR 相关蛋白9（Cas9）; 结节性硬化症; 基因敲除; 结节性硬化症1 基因
分类号:
R596.2；Q786
DOI:
10.3969/j.issn.1671-7414.2021.04.027
文献标志码:
A
摘要:
目的　利用成簇规律间隔短回文重复序列/ 成簇规律间隔短回文重复序列相关蛋白（clustered regularly interspacedshort palindromic repeat/CRISPR-associated protein，CRISPR/Cas9）基因编辑系统在HEK293 细胞系中对TSC1 基因稳定敲除，并对其敲除效果进行鉴定。方法　根据TSC1 基因的序列设计sgRNA，将sgRNA 克隆到载体lentilCRISPRv2 上，将连接产物转化至Stbl3 感受态细胞，对菌液进行测序鉴定。将鉴定成功的lentiCRISPRV2-sgTSC1 质粒转染至HEK293细胞，加入嘌呤霉素进行筛选，挑选单细胞克隆进行PCR 鉴定，选取条带单一的细胞株用Western blot 鉴定TSC1 基因的表达。结果　成功构建lentiCRISPRV2-sgTSC1-1/2 重组质粒，并利用该质粒完成对TCS1 基因的定点切割，Westernblot 结果显示细胞中无TSC1 表达。结论　用CRISPR/Cas9 系统成功构建TSC1 基因敲除的稳定细胞株，为后续实验奠定了基础。

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备注/Memo

备注/Memo:
作者简介：王晶（1993-），女，硕士研究生，检验技师，主要从事临床检验与分子生物学，E-mail:wjmed0429@163.com。
通讯作者：高倩，女，硕士研究生，主管技师，E-mail:1228225756@qq.com。
更新日期/Last Update: 1900-01-01