Expression of intracellular interferon-alpha confers antiviral properties in transfected bovine fetal fibroblasts and does not affect the full development of SCNT embryos.

Weihua Du,Jinxia Zhang,Huabin Zhu,Xueming Zhao,Zongxing Fan,Haisheng Hao,Yan Liu,Tong Qin,Shoufeng Zhang,Dawei Yu

doi:10.1371/journal.pone.0094444

Abstract

Foot-and-mouth disease, one of the most significant diseases of dairy herds, has substantial effects on farm economics, and currently, disease control measures are limited. In this study, we constructed a vector with a human interferon-α (hIFN-α) (without secretory signal sequence) gene cassette containing the immediate early promoter of human cytomegalovirus. Stably transfected bovine fetal fibroblasts were obtained by G418 selection, and hIFN-α transgenic embryos were produced by somatic cell nuclear transfer (SCNT). Forty-six transgenic embryos were transplanted into surrogate cows, and five cows (10.9%) became pregnant. Two male cloned calves were born. Expression of hIFN-α was detected in transfected bovine fetal fibroblasts, transgenic SCNT embryos, and different tissues from a transgenic SCNT calf at two days old. In transfected bovine fetal fibroblasts, expression of intracellular IFN-α induced resistance to vesicular stomatitis virus infection, increased apoptosis, and induced the expression of double-stranded RNA-activated protein kinase gene (PKR) and the 2′-5′-oligoadenylate synthetase gene (2′-5′ OAS), which are IFN-inducible genes with antiviral activity. Analysis by qRT-PCR showed that the mRNA expression levels of PKR, 2′-5′ OAS, and P53 were significantly increased in wild-type bovine fetal fibroblasts stimulated with extracellular recombinant human IFN-α-2b, showing that intracellular IFN-α induces biological functions similar to extracellular IFN-α. In conclusion, expression of intracellular hIFN-α conferred antiviral properties in transfected bovine fetal fibroblasts and did not significantly affect the full development of SCNT embryos. Thus, IFN-α transgenic technology may provide a revolutionary way to achieve elite breeding of livestock.

Highlights

Transgenic technology enables the introduction of exogenous genes into animal genomes and provides a revolutionary way to achieve elite breeding of livestock
Total protein extracts from bovine fetal fibroblasts were analyzed by western blotting using monoclonal antibodies against human interferon-a (hIFN-a)-2b and lactoferricin B (LFCIN B), with hIFN-a protein expression was detected in transfected bovine fetal fibroblasts (Fig. 2A, lane 3, 9), but not in extracts from wild-type bovine fetal fibroblasts (Fig. 2A, lanes 2, 8)
We focused on biological activities of IFN-a in transfected fetal fibroblasts and transgenic somatic cell nuclear transfer (SCNT) embryos

Summary

Introduction

Transgenic technology enables the introduction of exogenous genes into animal genomes and provides a revolutionary way to achieve elite breeding of livestock. Our efforts have focused on developing foot-and-mouth disease (FMD) resistance in dairy cattle using transgenic somatic cell nuclear transfer (SCNT) technology. Current vaccines and disease-control measures to eliminate FMD have many drawbacks [4] Several new strategies, such as RNA interference [5,6], have been developed to control FMD, but few reports have detailed transgenic livestock strategies [1]. A secretory signal sequence of IFN-a was deleted to verify whether intracellular expression of IFN-a has side effects in transgenic cattle. To produce transgenic SCNT cattle that can resist FMDV, we constructed a vector with a human IFN-a gene cassette containing the immediate early promoter of human cytomegalovirus (HCMV). Transgenic cattle containing IFN-a were born at full term

Materials and Methods

F: GAT GCG TCG TAT CTC TCT GTT C R

Results

Discussion