Please wait a minute...
Journal of Zhejiang University (Agriculture and Life Sciences)  2018, Vol. 44 Issue (2): 157-161    DOI: 10.3785/j.issn.1008-9209.2018.03.013
    
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

Download: HTML (   PDF(1959KB)
Export: BibTeX | EndNote (RIS)      

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 wordsCRISPR/Cas9n system      gene editing      blue ear disease      CD163 gene     
Published: 27 April 2018
Cite this article:

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.

URL:

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


利用CRISPR/Cas9n技术生产抗蓝耳病的基因编辑克隆猪

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 
No related articles found!