利用CRISPR/Cas9技术构建大鼠L2细胞α-ENaC基因敲除稳定细胞株

doi:10.13560/j.cnki.biotech.bull.1985.2019-0919

生物技术通报 ›› 2020, Vol. 36 ›› Issue (3): 115-123.doi: 10.13560/j.cnki.biotech.bull.1985.2019-0919

• 基因编辑专题（专题主编谷峰研究员） • 上一篇下一篇

利用CRISPR/Cas9技术构建大鼠L2细胞α-ENaC基因敲除稳定细胞株

王松¹, 李鹏程², 白朝辉³, 许宏鑫⁴, 应研敏⁵, 张墨³, 白义春¹

1. 新乡医学院公共卫生学院,新乡 453000;
2. 西北农林科技大学动物科技学院,杨凌712100;
3. 新乡医学院全过程教学基地,新乡 453000;
4. 新乡医学院第一临床学院,新乡 453000;
5. 新乡医学院法医学院,新乡 453000

收稿日期:2019-09-28 出版日期:2020-03-26 发布日期:2020-03-17
作者简介:王松,男,硕士研究生,研究方向：钠离子通道;E-mail：1252419529@qq.com
基金资助:
国家自然科学青年科学基金项目（31802024）

Generation of Stable α-ENaC Knockout Rat L2 Cell Strains by CRISPR/Cas9

WANG Song¹, LI Peng-cheng², BAI Chao-hui³, XU Hong-xin⁴, YING Yan-min⁵, ZHANG Mo³, BAI Yi-chun¹

1. School of Public Health,Xinxiang Medical University,Xinxiang 453000;
2. College of Animal Science and Technology,Northwest A&F University,Yangling 712100;
3. Whole Process Teaching Base,Xinxiang Medical University,Xinxiang 453000;
4. First Clinical College,Xinxiang Medical University,Xinxiang 453000;
5. School of Forensic Medicine,Xinxiang Medical University,Xinxiang 453000

Received:2019-09-28 Published:2020-03-26 Online:2020-03-17

摘要/Abstract

摘要：

利用CRISPR/Cas9基因编辑技术构建大鼠L2细胞α-ENaC基因敲除的细胞株,研究α-ENaC基因对细胞增殖的影响。构建敲除α-ENaC基因的CRISPR/Cas9表达载体和筛选报告载体,通过转染和嘌呤霉素筛选获得单克隆细胞株,Western Blot、测序确定突变的细胞株,CCK-8检测突变细胞株的增殖活力。成功构建靶向α-ENaC基因第一外显子的CRISPR/Cas9表达载体和筛选报告载体,嘌呤霉素筛选后,挑选8个单细胞克隆中有两个单细胞克隆α-ENaC蛋白表达下降,一个单细胞克隆α-ENaC蛋白不再表达,测序结果显示3个单细胞克隆分别为2个单等位基因突变和1个双等位基因突变,且未发现脱靶现象。突变细胞株的增殖活力降低,其中双等位基因突变细胞株增殖活力降低更为显著。因此,利用CRISPR/Cas9结合SSA-RPG报告载体成功获得了α-ENaC基因敲除的L2细胞株,α-ENaC与细胞增殖有关。

关键词: L2细胞, α-ENaC, CRISPR/Cas9, 基因敲除

Abstract:

The purposes of this study are to generate α-ENaC knockout rat L2 cell line using CRISPR/Cas9 gene engineering technology and to explore the effect of α-ENaC on cell proliferation. CRISPR/Cas9 expression vectors with the α-ENaC gene knockout and the surrogate reporter were constructed on rat L2 cell line. After transfection and puromycin selection,the obtained mutational monoclones were confirmed by Western Blot and sequence analysis. CCK-8 method was used to determine the proliferation activity of the mutated cells. The CRISPR/Cas9 systems targeting the first exon of α-ENaC gene and the SSA-RPG surrogate reporter were designed and constructed successfully. Eight monoclones were picked after puromycin selection,and 2 of 8 monoclones showed significantly reduced α-ENaC expression and one of them lost the α-ENaC expression. The sequence analysis results confirmed that 2 of 3 monoclones were one single allelic mutation and the other one was double allelic mutation. In addition,there were no off-targets mutations. The proliferation activity of mutant cell lines decreased significantly,and that of the double allelic mutant cell line decreased more significantly. In sum,the α-ENaC knockout rat L2 cell line is successfully generated using CRISPR/Cas9 system combined with SSA-RPG surrogate reporters,and the α-ENaC gene is related to cell proliferation.

Key words: L2 cell, α-ENaC, CRISPR/Cas9, gene knockout

王松, 李鹏程, 白朝辉, 许宏鑫, 应研敏, 张墨, 白义春. 利用CRISPR/Cas9技术构建大鼠L2细胞α-ENaC基因敲除稳定细胞株[J]. 生物技术通报, 2020, 36(3): 115-123.

WANG Song, LI Peng-cheng, BAI Chao-hui, XU Hong-xin, YING Yan-min, ZHANG Mo, BAI Yi-chun. Generation of Stable α-ENaC Knockout Rat L2 Cell Strains by CRISPR/Cas9[J]. Biotechnology Bulletin, 2020, 36(3): 115-123.

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利用CRISPR/Cas9技术构建大鼠L2细胞α-ENaC基因敲除稳定细胞株

Generation of Stable α-ENaC Knockout Rat L2 Cell Strains by CRISPR/Cas9

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