文献类型： 外文期刊
作者： Ishag, Hassan Z. A. 1 ; Liu, Maojun 1 ; Yang, Ruosong 1 ; Xiong, Qiyan 1 ; Feng, Zhixin 1 ; Shao, Guoqing 1 ;
作者机构： 1.Jiangsu Acad Agr Sci, Inst Vet Med, Natl Res Ctr Engn & Technol Vet Bioprod, Key Lab Vet Biol Engn & Technol,Minist Agr, Nanjing 210014, Peoples R China
2.Univ Nyala, Coll Vet Sci, Nyala, Sudan
关键词： eGFP;PK-15 Cells;NHEJ;HDR;CRISPR/Cas9
期刊名称：ROMANIAN BIOTECHNOLOGICAL LETTERS （ 影响因子：0.765； 五年影响因子：0.823 ）
ISSN： 1224-5984
年卷期： 2017 年 22 卷 4 期
页码：
收录情况： SCI
摘要： The aim of this study is to outline a simple method to efficiently disrupt eGFP gene in PK-15 cells using RNA-guided Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 system. We co-transfected a plasmid encoding both eGFP and it is sgRNA with a plasmid encoding Cas9 into PK-15 cells using Lipofectamine. After 24 hrs of culture, an about 65% of the cells had lost eGFP expression via non-homologous end joining (NHEJ), examined under the fluorescence microscope. When a donor plasmid carrying a blue fluorescence protein (BFP) with arms homologous to eGFP was introduced along with CRISPR plasmids, a BFP fluorescence replacing the green fluorescence of eGFP was observed, possibly induced by homology-directed repair (HDR). In conclusion, CRISPR/Cas9 system was successfully utilized in PK-15 cells to inactivate eGFP. This might be a useful technique to be developed to inactivate foreign genes, including invaders such as viruses and internal parasites reducing their burden.
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