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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

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.
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Fig3: 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.

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)

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

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4230515&req=5

Fig3: 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.
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