Production of transgenic pigs over-expressing the antiviral gene Mx1

Abstract

The myxovirus resistance gene (Mx1) has a broad spectrum of antiviral activities. It is therefore an interesting candidate gene to improve disease resistance in farm animals. In this study, we report the use of somatic cell nuclear transfer (SCNT) to produce transgenic pigs over-expressing the Mx1 gene. These transgenic pigs express approximately 15-25 times more Mx1 mRNA than non-transgenic pigs, and the protein level of Mx1 was also markedly enhanced. We challenged fibroblast cells isolated from the ear skin of transgenic and control pigs with influenza A virus and classical swine fever virus (CFSV). Indirect immunofluorescence assay (IFA) revealed a profound decrease of influenza A proliferation in Mx1 transgenic cells. Growth kinetics showed an approximately 10-fold reduction of viral copies in the transgenic cells compared to non-transgenic controls. Additionally, we found that the Mx1 transgenic cells were more resistant to CSFV infection in comparison to non-transgenic cells. These results demonstrate that the Mx1 transgene can protect against viral infection in cells of transgenic pigs and indicate that the Mx1 transgene can be harnessed to develop disease-resistant pigs.

Keywords: Antiviral breeding; Innate resistance; Somatic cell nuclear transfer.

Figure 1

Construction and expression of Mx1 transgenic vector. (A) Comparison of 2 alleles of Mx1 gene in Tibet miniature pigs (position 1675–1718 in nucleic acid sequence). The 3 base pair (bp)-deleted sequence is indicated with a dashed line. (B) Schematic diagram of the transgenic vector pMx1-2A-EGFP and the binding sites of primers used in genomic PCR assays to screen for the presence of the transgene are marked with arrows. The size of the PCR product using primers Mx1-2 and Mx1-7 is 192 bp. The same primers generate a PCR fragment of 575 bp from the endogenous genomic Mx1 due to the existence of an intron. The primer Mx1-5 is located in the 2A sequence and the transgenic vector produces a 750 bp DNA fragment when amplified by PCR using primers Mx1-1 and Mx1-5. These primers cannot amplify wild-type genomic DNA. (C) Schematic diagram of the Mx1 expression vector pVAX-Mx1. (D) Transient expression of pMx1-2A-EGFP and pVAX-Mx1 in 293 T cell. The arrowhead indicates the uncleaved transgenic Mx1-2A-EGFP protein. Arrow indicates the cleaved Mx1 protein.

Figure 2

Genotyping and expression analysis of Mx1 transgene in trangenic pigs. (A) PCR to identify piglets with genomic integration of the Mx1 transgene. Upper panel: PCR analysis with primers Mx1-2 and Mx1-7; Lower panel: PCR analysis with primers Mx1-1 and Mx1-5. The lanes are: M, marker; N, non-transgenic pig; #4-1, #4-2, #4-3, #4-4, #4-5, transgenic pigs; P, plasmid DNA. (B) Identification of mRNA level of Mx1 in the transgenic piglets using real time RT-PCR. Lanes 1–3 are naturally bred piglets, lanes 4–13 are cloned pigs without transgene integration, and lanes 14–18 are 5 cloned pigs that contained the transgene. Porcine GAPDH was used as reference control. Values represent the mean ± s.d. from triplicate experiments. Statistically significant P values are noted with an asterisk (*P < 0.001. One-way natural breeding pig was used to generate the P values). (C) Detecting the presence of Mx1 protein in fibroblasts of transgenic pigs by western blot. Cell lysates from HEK293T cells transiently transfected with pVAX-Mx1 were used as a positive control. (D) Expression of the Mx1 protein in various organs of transgenic and non-transgenic pigs. The arrowhead indicates the uncleaved transgenic Mx1-2A-EGFP protein and the arrow indicates the cleaved Mx1 protein.

Figure 3

Pictures of transgenic pigs and EGFP expression in the fibroblasts and hooves. (A) Picture of the 5 transgenic piglets taken at 1 month of age. (B) Expression of EGFP in ear fibroblasts isolated from a transgenic piglet; 20 × magnification. (C) Expression of EGFP in the hooves of a transgenic piglet.

Figure 4

Enhanced influenza A viral resistance in cultured fibroblasts isolated from transgenic pigs. (A) Ear fibroblasts isolated from Mx1 transgenic pigs and age-matched controls were infected with influenza A virus PR8 or NIBRG-14. After 24 hours, cells were fixed and stained for NP expression (red: NP, blue: nuclei; 4 × magnification). (B) Quantification of NP-positive cells. Values represent the mean ± s.d., n = 3. *P < 0.01 compared with the non-transgenic group.

Figure 5

Growth curves of influenza A viruses PR8 and NIBRG-14 in the isolated ear fibroblasts of Mx1 transgenic piglets. Growth curves of PR8 at a MOI of 1 (A) and 0.01 (B). Growth curves of NIBRG-14 at a MOI of 1 (C) and 0.01 (D). Values represent the mean ± s.d., n = 3.

Figure 6

Protective effect of Mx1 transgene against CSFV infection. (A) IFA and FITC conjugated antibodies were used to examine the viral infection in fibroblasts from 5 transgenic and an age-matched non-transgenic pigs; 4 × magnification. (B) Quantification of CSFV-positive cells. Values represent the mean ± s.d., n = 3. *P < 0.01 vs non-transgenic.