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
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
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