Generation of TALE nickase-mediated gene-targeted cows expressing human serum albumin in mammary glands.

Yan Luo,Qi Chen,Yong Zhang,Zekun Guo,Yongyan Wu,Xu Liu,Yongsheng Wang,Fusheng Quan,Jun Liu,Hui Lan,Chenchen Cui

doi:10.1038/srep20657

Abstract

Targeting exogenous genes at milk protein loci via gene-targeting technology is an ideal strategy for producing large quantities of pharmaceutical proteins. Transcription- activator-like effector (TALE) nucleases (TALENs) are an efficient genome-editing tool. However, the off-target effects may lead to unintended gene mutations. In this study, we constructed TALENs and TALE nickases directed against exon 2 of the bovine β-lactoglobulin (BLG) locus. The nickases can induce a site-specific DNA single-strand break, without inducing double-strand break and nonhomologous end joining mediated gene mutation, and lower cell apoptosis rate than TALENs. After co-transfecting the bovine fetal fibroblasts with human serum albumin (HSA) gene-targeting vector and TALE nickase expression vectors, approximately 4.8% (40/835) of the cell clones contained HSA at BLG locus. Unexpectedly, one homozygous gene-targeted cell clone (1/835, 0.1%) was obtained by targeting both alleles of BLG in a single round of transfection. The recombinant protein mimicking the endogenous BLG was highly expressed and correctly folded in the mammary glands of the targeted cows, and the expression level of HSA was significantly increased in the homozygous targeted cows. Results suggested that the combination of TALE nickase-mediated gene targeting and somatic cell nuclear transfer is a feasible and safe approach in producing gene-targeted livestock.

Highlights

Targeting exogenous genes at milk protein loci via gene-targeting technology is an ideal strategy for producing large quantities of pharmaceutical proteins
The results showed that both pairs of TALEN1/2 and TALEN3/4 restore GFP expression, indicating that TALENs can cleave the target bovine β-lactoglobulin (BLG) sequence, can trigger the non-homologous end joining (NHEJ) repair pathway, and can induce gene mutation
By using Transcription- activator-like effector (TALE) nickase combined with somatic cell nuclear transfer (SCNT), we obtained two homozygous and four heterozygous targeted cows that expressed high level of recombinant Human serum albumin (HSA) in their mammary glands

Summary

Introduction

Targeting exogenous genes at milk protein loci via gene-targeting technology is an ideal strategy for producing large quantities of pharmaceutical proteins. The nickases can induce a site-specific DNA single-strand break, without inducing double-strand break and nonhomologous end joining mediated gene mutation, and lower cell apoptosis rate than TALENs. After co-transfecting the bovine fetal fibroblasts with human serum albumin (HSA) gene-targeting vector and TALE nickase expression vectors, approximately 4.8% (40/835) of the cell clones contained HSA at BLG locus. Previous studies demonstrated that zinc-finger nickases (ZFNickases) derived from a ZFN pair via D450 mutation of the FokI catalytic domain can induce SSBs, and nickase-induced SSBs are sufficient to stimulate homology-directed repair (HDR) without NHEJ in human and animal cells[9,11,12] This finding suggested that nickase can improve the fidelity of gene modification. This study tested the feasibility of targeting HSA gene in exon 2 of the bovine BLG locus by TALE nickase and determined the efficiency of HSA expression in the milk of gene-targeted cows generated by somatic cell nuclear transfer (SCNT)

Methods

Results

Conclusion