Generation of buffalo mammary epithelial cells with targeted knockout of 18s rDNA by a CRISPR/Cas9 adenovirus system

文献类型： 外文期刊

第一作者： Zhu, Peng

作者： Zhu, Peng;Duan, An-Qin;Deng, Ting-Xian;Lu, Xing-Rong;Ma, Xiao-Ya;Liang, Sha-Sha;Pang, Chun-Ying;Liang, Xian-Wei;Zhu, Peng

作者机构：

关键词： CRISPR/Cas9;multiple-locus gene targeting;18s rDNA;buffalo;adenovirus

期刊名称：BIOTECHNOLOGY AND BIOPROCESS ENGINEERING （ 影响因子：2.836； 五年影响因子：2.281 ）

ISSN： 1226-8372

年卷期： 2017 年 22 卷 5 期

页码：

收录情况： SCI

摘要： In recent years, CRISPR/Cas9 has rapidly become one of the most promising genome editing tools because it is simple and easy to use and cost effective. However, the large size of Cas9 sequences limits its application in clinically promising vectors and it also impacts non-viral transfection. In this study, CRISPR/Cas9 adenovirus vectors that target the buffalo 18s rDNA gene were constructed, transfected into 293 cells for adenovirus packaging, and the adenovirus was used to knockout the 18s rDNA gene in buffalo mammary epithelial cells. The results demonstrated that the CRISPR/Cas9 adenovirus vectors for the buffalo 18s rDNA gene could efficiently target the sites as revealed by the fluorescence reporter system. After amplification, the adenovirus titer of Sn458-18s1 and Sn458-18s2 reached 1.03 x 10(9)PFU/mL and 1.05 x 10(9) PFU/mL, respectively. For buffalo mammary epithelial cell infection, the efficiency was 100% when the multiplicity of infection (MOI) ae 100 PFU/mL. There were 9 mutational clones found in the 20 clones, and the gene mutagenesis rate reached 45%. Of these, 2 clones were 35-bp deleted and 7 clones were 12-bp deleted. These results suggested that the adenovirus system overcame the low transfection efficiency of the buffalo mammary epithelial cells associated with using lipid-based methods or electroporation. Moreover, we preliminary developed an efficient technique for multiple-locus gene targeting at repeated sequences of the buffalo genome.

分类号：

  • 相关文献

[1]Generation of Genetically Modified Buffalo Embryos Using Multiple-Locus Gene Targeting. Pang, Chunying,Deng, Tingxian,Liang, Xianwei,Zhang, Yingyin,Yang, Bingzhuang. 2013

[2]The Construct of Fat-1 Gene Targeted Buffalo Kidney Fibroblast Cell. Zhang, Yingyin,Pang, Chunying,Deng, Tingxian,Yang, Bingzhuang,Liang, Xianwei. 2013

[3]Evaluation of protective immune response in mice by vaccination the recombinant adenovirus for expressing Schistosoma japonicum inhibitor apoptosis protein. Hu, Chao,Zhu, Lihui,Luo, Rong,Dao, Jinwei,Zhao, Jiangping,Shi, Yaojun,Li, Hao,Lu, Ke,Feng, Xingang,Lin, Jiaojiao,Liu, Jinming,Chen, Guofeng. 2014

[4]Relationship of long noncoding RNA and viruses. Zhang, Jie,Zhang, Yong-Guang.

[5]Phylogenetic analysis of Babesia species in China based on cytochrome b (COB) gene. Tian, Zhancheng,Luo, Jin,Zheng, Jinfeng,Xie, Junren,Shen, Hui,Yin, Hong,Luo, Jianxun,Tian, Meiyuan,Yuan, Xiaosong,Wang, Fangfang,Liu, Guangyuan. 2013

[6]Mudworm Polydora lingshuiensis sp n is a new species that inhabits both shell burrows and mudtubes. Ye, Lingtong,Tang, Bin,Su, Youlu,Wang, Ruixuan,Wang, Jiangyong,Ye, Lingtong,Tang, Bin,Su, Youlu,Wang, Ruixuan,Wang, Jiangyong,Ye, Lingtong,Tang, Bin,Su, Youlu,Wang, Ruixuan,Wang, Jiangyong,Ye, Lingtong,Yu, Ziniu,Tang, Bin,Wu, Kaichang.

[7]Khawia abbottinae sp n. (Cestoda: Caryophyllidea) from the Chinese false gudgeon Abbottina rivularis (Cyprinidae: Gobioninae) in China: morphological and molecular data. Xi, Bing Wen,Xie, Jun,Oros, Mikulas,Wang, Gui Tang,Scholz, Tomas.

[8]Genetic diversity and phylogeny of the family Osteoglossidae by the nuclear 18S ribosomal RNA and implications for its conservation. Mu, Xi-dong,Gu, Dang-en,Yang, Ye-xin,Luo, Du,Meng, Xu,Wang, Xue-jie,Hu, Yin-chang,Luo, Jian-ren.

[9]Marine fungal communities in water and surface sediment of a sea cucumber farming system: habitat-differentiated distribution and nutrients driving succession. Guo, Xiaohong,Zhang, Qianqian,Zhang, Xiaoli,Gong, Jun,Guo, Xiaohong,Zhang, Jiasong.

[10]First report on the occurrence of Theileria sp OT3 in China. Tian, Zhancheng,Liu, Guangyuan,Yin, Hong,Xie, Junren,Wang, Suyan,Yuan, Xiaosong,Wang, Fangfang,Luo, Jin. 2014

[11]Rapid detection and identification of four major Schistosoma species by high-resolution melt (HRM) analysis. Li, Juan,Zhu, Xing-Quan,Li, Juan,Zhao, Guang-Hui,Lin, RuiQing,Blair, David,Sugiyama, Hiromu.

[12]The effective expression of xylanase gene in Candida utilis by 18S rDNA targeted homologous recombination in pGLR9K. Wang Wei,Yang Hong-Lan,Bao HuiFang,Shan Qi-mu-ge,Yang Hong-Lan,Zhang Daoyuan,Wang Wei,Shan Qi-mu-ge,Wood, Andrew J..

[13]The Impact of Linolenic Acid on in vitro Development of Buffalo Embryos. Zheng, Haiying,Yang, Chunyan,Shang, Jianghua,Pang, Chunying,Huang, Fenxiang,Wang, Jian,Yang, Bingzhuang,Liang, Xianwei. 2013

[14]Optimization of Culture and Cryopreservation of Hand-Made Clone (HMC) Buffalo (Bubalus bubalis) Embryos. Yang, Chunyan,Pang, Chunying,Zheng, Haiying,Huang, Fenxiang,Wang, Jian,Shang, Jianghua,Yang, Bingzhuang,Liang, Xianwei. 2013

[15]Repeated Ovum Pick-Up and In-vitro Embryo Production in Buffalo. Liang, X.,Zhang, X.,Yang, B.,Chen, M.,Huang, F.,Pang, C.,Liao, C.,Qin, G.,Wei, S.,Yang, C.. 2007

[16]Milk fat globule is an alternative to mammary epithelial cells for gene expression analysis in buffalo. Chen, Qiuming,Wu, Yanjun,Zhang, Mingyuan,Xu, Wenwen,Guo, Xiaoping,Yan, Xueyu,Deng, Haiying,Jiang, Qinyang,Yang, Xiurong,Lan, Ganqiu,Guo, Yafen,Jiang, Hesheng,Qin, Guangsheng.

[17]A field study on artificial insemination of swamp and crossbred buffaloes with sexed semen from river buffaloes. Lu, Yangqing,Liao, Yanqiong,Zhang, Ming,Yang, Xiaogan,Lu, Shengsheng,Xu, Huiyan,Lu, Kehuan,Liao, Yanqiong,Yang, Bingzhuang,Liang, Xianwei,Wu, Zhuyue,Liang, Yunbin.

[18]Four novel polymorphisms of buffalo INSIG2 gene are associated with milk production traits in Chinese buffaloes. Deng, Tingxian,Pang, Chunying,Ma, Xiaoya,Lu, Xingrong,Duan, Anqin,Zhu, Peng,Liang, Xianwei.

[19]Effects of Curcumin on Buffalo Embryonic Development In Vitro. Shang, Jiang-Hua,Yang, Chun-Yan,Zheng, Hai-Ying,Jing-Qin,Wang, Jian,Huang, Fen-Xiang,Yang, Bing-Zhuang,Liang, Xiang-Wei. 2013

[20]Associations between polymorphisms of the STAT1 gene and milk production traits in water buffaloes. Deng, T. X.,Pang, C. Y.,Lu, X. R.,Zhu, P.,Duan, A. Q.,Liang, X. W..

作者其他论文 更多>>

微信小程序