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Production of transgenic pigs over-expressing the antiviral gene Mx1.

Yan Q, Yang H, Yang D, Zhao B, Ouyang Z, Liu Z, Fan N, Ouyang H, Gu W, Lai L - Cell Regen (Lond) (2014)

Bottom Line: It is therefore an interesting candidate gene to improve disease resistance in farm animals.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.

View Article: PubMed Central - PubMed

Affiliation: Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, China.

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.

No MeSH data available.


Related in: MedlinePlus

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.
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Fig2: 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.

Mentions: PCR analysis of genomic DNA of each piglet showed that 5 piglets were positive for the Mx1 transgene and all of which had been derived from transgenic cell colony #4 (Figure 2A). Real time RT-PCR analysis revealed that these transgenic piglets carried 5 copies of the transgene. Additionally, we determined that Mx1 mRNA levels in fibroblasts isolated from the 5 transgenic piglets were 15–25 times higher than in cloned piglets lacking the transgene as well as in naturally bred piglets (Figure 2B). Western blot showed enhanced Mx1 protein levels in fibroblasts isolated from transgenic pigs (Figure 2C). Mx1 protein levels were also elevated in the organs (heart, lung, liver, muscle) of the deceased transgenic pig #4-4 as compared to non-transgenic pigs (Figure 2D). We also observed EGFP expression in fibroblasts and hooves of all 5 transgenic piglets further illustrating the successful generation of transgenic animals that robustly express the Mx1 gene in a wide variety of tissues (Figure 3A, B, C).Table 1


Production of transgenic pigs over-expressing the antiviral gene Mx1.

Yan Q, Yang H, Yang D, Zhao B, Ouyang Z, Liu Z, Fan N, Ouyang H, Gu W, Lai L - Cell Regen (Lond) (2014)

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.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4230515&req=5

Fig2: 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.
Mentions: PCR analysis of genomic DNA of each piglet showed that 5 piglets were positive for the Mx1 transgene and all of which had been derived from transgenic cell colony #4 (Figure 2A). Real time RT-PCR analysis revealed that these transgenic piglets carried 5 copies of the transgene. Additionally, we determined that Mx1 mRNA levels in fibroblasts isolated from the 5 transgenic piglets were 15–25 times higher than in cloned piglets lacking the transgene as well as in naturally bred piglets (Figure 2B). Western blot showed enhanced Mx1 protein levels in fibroblasts isolated from transgenic pigs (Figure 2C). Mx1 protein levels were also elevated in the organs (heart, lung, liver, muscle) of the deceased transgenic pig #4-4 as compared to non-transgenic pigs (Figure 2D). We also observed EGFP expression in fibroblasts and hooves of all 5 transgenic piglets further illustrating the successful generation of transgenic animals that robustly express the Mx1 gene in a wide variety of tissues (Figure 3A, B, C).Table 1

Bottom Line: It is therefore an interesting candidate gene to improve disease resistance in farm animals.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.

View Article: PubMed Central - PubMed

Affiliation: Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, China.

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.

No MeSH data available.


Related in: MedlinePlus