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

Qingyong Meng,Fei Liu,Zhaolin Sun,Fei Chang,Ning Li,Yaofeng Zhao,Ying Guo,Zhengquan Yu,Jing Fei,Yunping Dai,Mengxu Ge

doi:10.15302/j-fase-2015059

Abstract

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.

Highlights

Lactoferrin (LF) is a multifunctional iron binding glycoprotein belonging to the transferrin family, with a molecular weight of 80 kDa
The basic promoter of the mouse lactoferrin gene is located between nucleotides – 234 and – 21; deletion of this region completely abolishes its activity[25]
The reporter-transfected cells will express only monomeric red fluorescent protein (mRFP) in the absence of human codon-optimized SpCas9 (hSpCas9) nuclease activity; in the presence of nuclease activity, the transfected cells will express both mRFP and enhanced GFP (eGFP)[26]

Summary

Introduction

Lactoferrin (LF) is a multifunctional iron binding glycoprotein belonging to the transferrin family, with a molecular weight of 80 kDa. In 1938, LF was first identified as a red protein that was present in bovine milk[1]. In 1960, it was isolated from human milk by Groves and was subsequently recognized as a major iron binding protein[2,3,4]. Of the next-generation genome editing technologies, the CRISPR (clustered regularly interspaced short palindromic repeats)/Cas (CRISPR-associated) system has been demonstrated to be a robust tool for genome engineering. The type II CRISPR/Cas system has been demonstrated to be an efficient gene-editing technology that has been used successfully to produce many gene-targeted cell lines and animals[19,20,21,22,23,24]

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