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浙江大学学报(农业与生命科学版)  2018, Vol. 44 Issue (2): 157-161    DOI: 10.3785/j.issn.1008-9209.2018.03.013
论文     
利用CRISPR/Cas9n技术生产抗蓝耳病的基因编辑克隆猪
王少华1,赵盼盼1,刘通1,丁彪2,罗磊2,曹祖兵2,张运海2,张坤1*
1.浙江大学动物科学学院,哺乳动物分子胚胎学实验室,杭州 310058;2. 安徽农业大学动物科技学院,合肥 230036
Production of porcine reproductive and respiratory syndrome virus (PRRSV)-resistant genomeedited cloned pigs using CRISPR/Cas9n system
WANG Shaohua1, ZHAO Panpan1, LIU Tong1, DING Biao2, LUO Lei2, CAO Zubing2, ZHANG Yunhai2, ZHANG Kun1*

1. Laboratory of Mammalian Molecular Embryology, College of Animal Sciences, Zhejiang University, Hangzhou 310058, China; 2. College of Animal Science and Technology, Anhui Agricultural University, Hefei 230036, China

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摘要:

本研究利用基于CRISPR/Cas9n系统的基因编辑技术结合体细胞核移植技术,构建了在同一载体中同时表达2条sgRNA 和Cas9切刻酶以及绿色荧光蛋白(green fluorescent protein, GFP)的质粒,瞬时转染猪的成纤维细胞。通过流式细胞仪分选获得表达GFP的细胞,继续培养GFP阳性细胞至单个细胞长成细胞克隆点,经聚合酶链式反应(polymerase chain reaction, PCR)和DNA测序鉴定获得了对CD163基因进行编辑的细胞克隆点,阳性率高达90%(18/20)。以CD163基因编辑的细胞为供体细胞进行体细胞克隆和胚胎移植,获得了2头正常存活的CD163基因编辑猪。

关键词: CRISPR/Cas9n系统基因编辑蓝耳病CD163基因    
Abstract:

Using gene editing technology based on CRISPR/Cas9n system and somatic cell nuclear transfer technology, we constructed a plasmid of two sgRNAs and Cas9 nickase, and green fluorescent protein (GFP) in the same vector followed by transient transfection of porcine fibroblasts. By flow cytometry, the cells expressing GFP were selected, and the GFP positive cells were cultured to single cells. The CD163 edited clones were screened through polymerase chain reaction (PCR) and DNA sequencing, and the positive rate was as high as 90% (18/20). Somatic cell nuclear transfer and embryo transfer were carried out using cells with edited CD163, and two CD163 gene-edited cloned offsprings were obtained.

Key words: CRISPR/Cas9n system    gene editing    blue ear disease    CD163 gene
出版日期: 2018-04-27
基金资助: 国家自然科学基金面上项目(31672416);浙江大学“百人计划”启动经费
通讯作者: 张坤(https://orcid.org/0000-0002-2324-9381)     E-mail: kzhang@zju.edu.cn
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引用本文:

王少华, 赵盼盼, 刘通, 丁彪, 罗磊, 曹祖兵, 张运海, 张坤. 利用CRISPR/Cas9n技术生产抗蓝耳病的基因编辑克隆猪[J]. 浙江大学学报(农业与生命科学版), 2018, 44(2): 157-161.

WANG Shaohua, ZHAO Panpan, LIU Tong, DING Biao, LUO Lei, CAO Zubing, ZHANG Yunhai, ZHANG Kun. Production of porcine reproductive and respiratory syndrome virus (PRRSV)-resistant genomeedited cloned pigs using CRISPR/Cas9n system. Journal of Zhejiang University (Agriculture and Life Sciences), 2018, 44(2): 157-161.

链接本文:

http://www.zjujournals.com/agr/CN/10.3785/j.issn.1008-9209.2018.03.013        http://www.zjujournals.com/agr/CN/Y2018/V44/I2/157

[1] 舒心媛, 严旭, 蒲烨弘, 王超, 潘建伟. CRISPR/Cas系统的作用原理及其在作物遗传改良中的应用[J]. 浙江大学学报(农业与生命科学版), 2018, 44(3): 259-268.