上海交通大学学报（医学版）

上海交通大学学报（医学版） >

2021 , Vol. 41 >Issue 3: 297 - 301

DOI: https://doi.org/10.3969/j.issn.1674-8115.2021.03.002

论著·基础研究

构建诱导型CRISPR/Cas9系统用于小鼠免疫细胞基因功能研究

  • 赵艳娜 ,
  • 邱荣 ,
  • 沈南 ,
  • 唐元家
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  • 1.上海交通大学医学院附属仁济医院风湿病科，上海市风湿病学研究所，上海 200127
    2.中国科学院上海营养与健康研究所，上海 200031
赵艳娜（1997—），女，硕士生；电子信箱：nanalr0704@163.com|邱荣（1989—），男，博士生；电子信箱：rqiu@sibs.ac.cn。

收稿日期: 2020-03-11

  网络出版日期: 2021-04-06

基金资助

国家自然科学基金(81871287);上海交通大学医学院高水平地方高校创新团队(SSMU-ZDCX20180100)

收起

Construction of inducible CRISPR/Cas9 system for studying gene function in mouse

  • Yan-na ZHAO ,
  • Rong QIU ,
  • Nan SHEN ,
  • Yuan-jia TANG
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  • 1.Shanghai Institute of Rheumatology, Department of Rheumatology, Renji Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200127, China
    2.Shanghai Institute of Nutrition and Health, Chinese Academy of Sciences, Shanghai 200031, China

Received date: 2020-03-11

  Online published: 2021-04-06

Supported by

National Natural Science Foundation of China(81871287);Innovative Research Team of High-Level Local Universities in Shanghai(SSMU-ZDCX20180100)

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摘要

目的·结合Dox诱导型单链导向RNA（single guide RNA，sgRNA）表达载体和Cas9转基因小鼠，构建诱导型CRISPR/Cas9系统用于小鼠免疫细胞基因功能研究。方法·根据四环素诱导表达系统原理，基因合成U6-TetO-sgRNA和EF1α-T2A-Puro-BFP-T2A-TetR片段。通过同源重组将2个片段组装进反转录病毒载体骨架，获得Dox诱导型sgRNA反转录病毒载体。为了验证系统有效性，分离Cas9转基因小鼠骨髓细胞并诱导其向巨噬细胞方向分化。设计对照 （non-targeting control，NC）组和实验组（靶向F4/80） 的sgRNA，利用反转录病毒感染细胞，分化条件设置添加Dox组（Dox+）和不添加Dox组（Dox-）。通过流式细胞术和T7核酸内切酶Ⅰ（T7 endonuclease Ⅰ，T7EⅠ）实验检测基因敲除效果。结果·①成功构建Dox诱导型sgRNA反转录病毒表达载体和Cas9转基因小鼠。②流式结果显示，在NC Dox-组、NCDox+组和F4/80 Dox-组中，几乎无F4/80阴性细胞群体；而在F4/80Dox+组中，F4/80阴性细胞群体高达50%。③T7EⅠ结果显示，在F4/80 Dox-组中，DNA条带完整，而在F4/80 Dox+组中发生基因突变，DNA条带被切开。结论·结合Dox诱导型sgRNA表达载体和Cas9转基因小鼠，成功构建诱导型CRISPR/Cas9系统。利用该系统成功在小鼠免疫细胞中实现可诱导性基因敲除。

关键词： 诱导型CRISPR/Cas9系统; Dox 诱导型sgRNA表达载体; 造血干细胞; 基因编辑; 巨噬细胞

本文引用格式

赵艳娜 , 邱荣 , 沈南 , 唐元家 . 构建诱导型CRISPR/Cas9系统用于小鼠免疫细胞基因功能研究[J]. 上海交通大学学报（医学版）, 2021 , 41(3) : 297 -301 . DOI: 10.3969/j.issn.1674-8115.2021.03.002

Abstract

Objective

·To construct inducible CRISPR/Cas9 system for studying gene function in mouse immune cells, combining Dox-inducible single guide RNA (sgRNA) expression vector with Cas9 transgenic mice.

Methods

·U6-TetO-sgRNA and EF1α-T2A-Puro-BFP-T2A-TetR fragments were obtained by gene synthesis. The two synthetic fragments were assembled into the retroviral vector backbone by using homologous recombination. sgRNA targeting protein coding region of F4/80 and non-targeting control (NC) were designed. Bone marrow cells were isolated from Cas9 transgenic mice and transfected with retrovirus expressing sgRNA. The experimental conditions were divided into Dox-added group (Dox +) and non Dox-added group (Dox-). The knockout effect was tested by flow cytometry and T7 endonuclease Ⅰ （T7EⅠ) experiments.

Results

·①Dox-inducible sgRNA retroviral vector and Cas9 transgenic mice were successfully constructed. ② The result of flow cytometry showed that F4/80 was only knocked out in the F4/80Dox+ population, but not in NC Dox-, NC Dox+ and F4/80 Dox- populations. ③ T7EⅠ results showed that the DNA was cut into two bands in the F4/80Dox+ group, while the DNA band was intact in the F4/80 Dox- group.

Conclusion

·An inducible CRISPR/Cas9 system combining Dox-inducible sgRNA retroviral vector with Cas9 transgenic mice are successfully constructed. With this system, inducible gene knockout in mouse immune cells are successfully achieved.

Key words： inducible CRISPR/Cas9 system; Dox-inducible sgRNA expression vector; hematopoietic stem cell; gene editing; macrophage

参考文献

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