Identification of Glyceraldehyde-3-Phosphate Dehydrogenase Gene as an Alternative Safe Harbor Locus in Pig Genome.

Xiaosong Han,Jinxue Ruan,Shuhong Zhao,Xiangdong Liu,Xinyun Li,Changzhi Zhao,Shengsong Xie,Kui Li,Changchun Li,Youcai Xiong

doi:10.3390/genes10090660

Abstract

The ectopic overexpression of foreign genes in animal genomes is an important strategy for gain-of-function study and establishment of transgenic animal models. Previous studies showed that two loci (Rosa26 and pH11) were identified as safe harbor locus in pig genomes, which means foreign genes can be integrated into this locus for stable expression. Moreover, integration of a transgene may interfere with the endogenous gene expression of the target locus after the foreign fragments are inserted. Here, we provide a new strategy for efficient transgene knock-in in the endogenous GAPDH gene via CRISPR/Cas9 mediated homologous recombination. This strategy has no influence on the expression of the endogenous GAPDH gene. Thus, the GAPDH locus is a new alternative safe harbor locus in the pig genome for foreign gene knock-ins. This strategy is promising for agricultural breeding and biomedical model applications.

Highlights

Integration of exogenous genes into the genome for stable expression is an important strategy for gain-of-function study and establishment of transgenic animal models
We investigated if house-keeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH) can be used as an alternative safe harbor locus
This study aims to identify whether the endogenous GAPDH gene can act as an alternative safe harbor locus in the pig genome

Summary

Introduction

Integration of exogenous genes into the genome for stable expression is an important strategy for gain-of-function study and establishment of transgenic animal models. Random integration of foreign genes often leads to unstable expression or unpredictable phenotypes [4]. Homologous recombination (HR) is an important pathway involved in the repair of double-strand DNA breaks [5], and HR-mediated gene knock-in allows the precise insertion of foreign fragments. HR-mediated integration in site-specific locus was used in the past to produce animal models [6,7]. This method is inefficient because of low efficiency [8], and laborious as construction of donor vectors is required

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