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Generation of CRISPR/Cas9-mediated lactoferrin-targeted mice by pronuclear injection of plasmid pX330

Mengxu GE,Fei LIU,Fei CHANG,Zhaolin SUN,Jing FEI,Ying GUO,Yunping DAI,Zhengquan YU,Yaofeng ZHAO,Ning LI,Qingyong MENG

《农业科学与工程前沿（英文）》 2015年 第2卷 第3期   页码 242-248 doi: 10.15302/J-FASE-2015059

摘要： Lactoferrin is a member of the transferrin family of multifunctional iron binding glycoproteins. While numerous physiological functions have been described for lactoferrin, the mechanisms underlying these functions are not clear. To further study the functions and mechanisms of lactoferrin, we modified the lactoferrin promoter of mice using the CRISPR/Cas9 system to reduce or eliminate lactoferrin expression. Seven mice with lactoferrin promoter mutations were obtained with an efficiency of 24% (7/29) by injecting the plasmid pX330, expressing a small guide RNA and human codon-optimized SpCas9, into fertilized eggs of mice. Plasmid integration and off-targeting of pX330 were not detected. These results confirmed that pronuclear injection of a circular plasmid is a feasible and efficient method for targeted mutagenesis in mice.

关键词： lactoferrin     promoter     CRISPR/Cas9     plasmid pX330    

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Expression of recombinant human butyrylcholinesterase in the milk of transgenic mice

Dan LU,Shengzhe SHANG,Shen LIU,Ying WU,Fangfang WU,Tan TAN,Qiuyan LI,Yunping DAI,Xiaoxiang HU,Yaofeng ZHAO,Ning LI

《农业科学与工程前沿（英文）》 2014年 第1卷 第3期   页码 179-184 doi: 10.15302/J-FASE-2014020

摘要： Butyrylcholinesterase (BCHE) is a natural bioscavenger that protects humans against organophosphate toxicity. Due to the limited yield of human BCHE (hBCHE) when purifying from human plasma, it is necessary to find an alternative method to produce this protein. One potential method is to produce transgenic livestock that make modified milk containing high concentration of hBCHE. In this study, we cloned the gene into a human lactoferrin (hLF) bacterial artificial chromosome (BAC) construct to make a hLF-hBCHE BAC construct. Subsequently, we injected the BAC construct into pronuclei of mouse fertilized embryos and generated transgenic mice. Expression analysis showed that recombinant hBCHE (rhBCHE) was expressed efficiently in the mammary gland of the transgenic mice and the concentration of rhBCHE in the milk of individual mice ranged from 76±12 to 159±28 mg·L . Protein function tests showed that rhBCHE has the same enzymatic activity as the native hBCHE. Our results pave the way for making transgenic livestock to produce large quantities of rhBCHE.

关键词： recombinant human butyrylcholinesterase (rhBCHE)     human lactoferrin bacterial artificial chromosome (hLF BAC)     transgenic mice     milk    

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标题 作者 时间 类型 操作

Generation of CRISPR/Cas9-mediated lactoferrin-targeted mice by pronuclear injection of plasmid pX330

Mengxu GE,Fei LIU,Fei CHANG,Zhaolin SUN,Jing FEI,Ying GUO,Yunping DAI,Zhengquan YU,Yaofeng ZHAO,Ning LI,Qingyong MENG

期刊论文

Expression of recombinant human butyrylcholinesterase in the milk of transgenic mice

Dan LU,Shengzhe SHANG,Shen LIU,Ying WU,Fangfang WU,Tan TAN,Qiuyan LI,Yunping DAI,Xiaoxiang HU,Yaofeng ZHAO,Ning LI

期刊论文