Expression of Intracellular Interferon-Alpha Confers Antiviral Properties in Transfected Bovine Fetal Fibroblasts and Does Not Affect the Full Development of SCNT Embryos

PLOS ONE, Dec 2019

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.

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Expression of Intracellular Interferon-Alpha Confers Antiviral Properties in Transfected Bovine Fetal Fibroblasts and Does Not Affect the Full Development of SCNT Embryos

et al. (2014) Expression of Intracellular Interferon-Alpha Confers Antiviral Properties in Transfected Bovine Fetal Fibroblasts and Does Not Affect the Full Development of SCNT Embryos. PLoS ONE 9(7): e94444. doi:10.1371/journal.pone.0094444 Expression of Intracellular Interferon-Alpha Confers Antiviral Properties in Transfected Bovine Fetal Fibroblasts and Does Not Affect the Full Development of SCNT Embryos Dawei Yu 0 Shoufeng Zhang 0 Weihua Du 0 Jinxia Zhang 0 Zongxing Fan 0 Haisheng Hao 0 Yan Liu 0 Xueming Zhao 0 Tong Qin 0 Huabin Zhu 0 Glenn Francis Browning, The University of Melbourne, Australia 0 1 Embryo Biotechnology and Reproduction Laboratory, Institute of Animal Science, Chinese Academy of Agricultural Sciences , Beijing , China , 2 Institute of Military Veterinary, Academy of Military Medical Science , Changchun , China , 3 State Key Laboratories of Agrobiotechnology, College of Biological Science, China Agricultural University , Beijing , China 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-a (hIFN-a) (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-a 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-a 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-a induced resistance to vesicular stomatitis virus infection, increased apoptosis, and induced the expression of double-stranded RNA-activated protein kinase gene (PKR) and the 29-59oligoadenylate synthetase gene (29-59 OAS), which are IFN-inducible genes with antiviral activity. Analysis by qRT-PCR showed that the mRNA expression levels of PKR, 29-59 OAS, and P53 were significantly increased in wild-type bovine fetal fibroblasts stimulated with extracellular recombinant human IFN-a-2b, showing that intracellular IFN-a induces biological functions similar to extracellular IFN-a. In conclusion, expression of intracellular hIFN-a conferred antiviral properties in transfected bovine fetal fibroblasts and did not significantly affect the full development of SCNT embryos. Thus, IFN-a transgenic technology may provide a revolutionary way to achieve elite breeding of livestock. - Funding: This work was partially supported by grants from the Agricultural Science and Technology Innovation Program (ASTIP-IAS06 to WHD), State Major Project of Transgenic (2009ZX08007-007B to WHD). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. Transgenic technology enables the introduction of exogenous genes into animal genomes and provides a revolutionary way to achieve elite breeding of livestock. The main applications of transgenic technology in livestock breeding include improving their disease resistance, carcass composition, lactational performance, wool production, growth rate, and reproductive performance, as well as reducing their environmental impact [1]. Our efforts have focused on developing foot-and-mouth disease (FMD) resistance in dairy cattle using transgenic somatic cell nuclear transfer (SCNT) technology. FMD is a highly contagious vesicular disease of cloven-hoofed animals [2]. Outbreaks of FMD can have severe economic and social consequences that result in the loss of billions of dollars ($US) in direct and indirect costs, as well as the slaughter of millions of animals [3]. 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]. Evidence suggests that expression of exogenous IFN-a in livestock confers resistance to FMDV infection [2,7]. Interferons (IFNs) are widely expressed cytokines that have potent antiviral and growth-inhibitory effects; they are the first line of defense against virus infections [8,9]. However, several reports indicate that side effects are associated with over-expression of secreted IFN-a in animal models, such as disrupted spermatogenesis in male transgenic mice [10,11]. In this study, cloned transgenic cattle containing IFN-a were generated to produce FMDV-resistant cattle. A secretory signal sequence of IFN-a was deleted to verify whether intracellular expression of IFN-a has side effects in (...truncated)


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Dawei Yu, Shoufeng Zhang, Weihua Du, Jinxia Zhang, Zongxing Fan, Haisheng Hao, Yan Liu, Xueming Zhao, Tong Qin, Huabin Zhu. Expression of Intracellular Interferon-Alpha Confers Antiviral Properties in Transfected Bovine Fetal Fibroblasts and Does Not Affect the Full Development of SCNT Embryos, PLOS ONE, 2014, Volume 9, Issue 7, DOI: 10.1371/journal.pone.0094444