CRISPR/Cas9系统介导的miR-155基因敲除细胞的制备

doi:10.13560/j.cnki.biotech.bull.1985.2019-0609

生物技术通报 ›› 2019, Vol. 35 ›› Issue (11): 231-239.doi: 10.13560/j.cnki.biotech.bull.1985.2019-0609

CRISPR/Cas9系统介导的miR-155基因敲除细胞的制备

李聪聪^{1, 2, 3}, 张永辉¹, 赵婉霞¹, 吴姣^{1, 2, 3}, 韩浩园^{1, 2, 3}, 李梦云¹, 牛晖^{1, 2, 3}, 宋素芳^{1, 2, 3}, 李婉涛^{1, 2, 3}

1. 河南牧业经济学院动物科技学院,郑州 450046;
2. 河南省畜禽遗传资源保护工程技术研究中心,郑州 450046;
3. 郑州市动物生殖分子调控重点实验室,郑州 450046

收稿日期:2019-07-08 出版日期:2019-11-26 发布日期:2019-11-19
作者简介:李聪聪,女,博士,讲师,研究方向：分子生物学与育种;E-mail：congcong_925520@126.com
基金资助:
国家自然科学基金项目（31702102）,河南省高等学校重点科研项目（18A230002）,河南牧业经济学院博士启动资金项目（53000139）,郑州市1125创新领军人才项目（2016XL003）

Preparation of miR-155 Knockout Cells Mediated by CRISPR/Cas9 Technology

LI Cong-cong^{1, 2, 3}, ZHANG Yong-hui¹, ZHAO Wan-xia¹, WU Jiao^{1, 2, 3}, HAN Hao-yuan^{1, 2, 3}, LI Meng-yun¹, NIU Hui^{1, 2, 3}, SONG Su-fang^{1, 2, 3}, LI Wan-tao^{1, 2, 3}

1. College of Animal Science and Technology,Henan University of Animal Husbandry and Economy,Zhengzhou 450046;
2. Henan Livestock and Poultry Genetic Resources Protection Engineering Technology Research Center,Zhengzhou 450046;
3. Zhengzhou Key Laboratory of Animal Reproduction Molecular Regulation,Zhengzhou 450046

Received:2019-07-08 Published:2019-11-26 Online:2019-11-19

摘要/Abstract

摘要： 为研究miR-155基因在猪肺泡巨噬细胞系（3D4/21）中的功能,采用CRISPR/Cas9系统构建miR-155敲除的猪肺泡巨噬细胞系。首先,采用sgRNA-cas9程序,设计4个靶向miR-155的特异性sgRNA引物序列。然后构建sgRNA表达载体,与pEGFP-C1-cas9载体共转染3D4/21细胞,进行流式分选并富集表达绿色荧光蛋白GFP的细胞。最后,采用T7E I酶酶切、Sanger测序、实时荧光定量PCR（qPCR）、western blot等方法检测敲除效率,发现miR-155基因可以被以上4个sgRNA编辑,其中sgRNA39的敲除效率最高,T7E I酶酶切检测流式分选后的敲除效率有42%;Sanger测序显示sgRNA39细胞系敲除31个碱基;RT-qPCR 结果显示miR-155-5p/3p表达量均有下降;western blot结果表明miR-155靶基因SHIP1的蛋白表达量升高。成功构建miR-155敲除的3D4/21细胞为进一步探讨miR-155在巨噬细胞发挥的调控功能研究提供细胞模型。

关键词: CRISPR/Cas9, miR-155, 敲除, 猪肺泡巨噬细胞

Abstract: To study the function of miR-155 in porcine alveolar macrophage cell line（3D4/21）,miR-155 knockout cells were constructed using CRISPR/Cas9 technology. Firstly,4 prime series specific to sgRNAs for targeting miR-155 were designed using sgRNA-cas9 program. Then,sgRNA expression vectors were constructed and co-transfected with pEGFP-C1-cas9 vector into 3D4/21 cells. Next,the cells expressing green fluorescent protein（GFP）were enriched and selected using the FACS-sorting strategy. Finally,T7E I cleavage assay,Sanger sequencing,real-time quantitative PCR（RT-qPCR）and Western blot were used to detect the efficiency of the knockout. We found that miR-155’s genomic sequence was edited using 4 different sgRNAs,while the highest efficiency of knockout reached by sgRNA39,up to 42% after T7EI enzyme digestion and FACS-sorting. Sanger sequencing demonstrated that 31 nucleotides of sgRNA39 cell line were knocked out. The RT-qPCR result showed that the expression levels of miR-155-5p/3p significantly decreased. The Western blot result indicated that the protein expression level of target gene SHIP1 in miR-155 significantly increased. To sum up,the successful establishment of the miR-155 knockout cell line provides cell model for further studying the regulation function of miR-155 in macrophage cells.

Key words: CRISPR/Cas9, miR-155, knockout, porcine alveolar macrophage cell line

李聪聪, 张永辉, 赵婉霞, 吴姣, 韩浩园, 李梦云, 牛晖, 宋素芳, 李婉涛. CRISPR/Cas9系统介导的miR-155基因敲除细胞的制备[J]. 生物技术通报, 2019, 35(11): 231-239.

LI Cong-cong, ZHANG Yong-hui, ZHAO Wan-xia, WU Jiao, HAN Hao-yuan, LI Meng-yun, NIU Hui, SONG Su-fang, LI Wan-tao. Preparation of miR-155 Knockout Cells Mediated by CRISPR/Cas9 Technology[J]. Biotechnology Bulletin, 2019, 35(11): 231-239.

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CRISPR/Cas9系统介导的miR-155基因敲除细胞的制备

Preparation of miR-155 Knockout Cells Mediated by CRISPR/Cas9 Technology

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