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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

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.
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Fig5: 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.

Mentions: Next we asked whether high expression levels of Mx1 rendered transgenic cells resistant to influenza A infection. To test this proposition, ear fibroblasts from transgenic pigs as well as non-transgenic controls were isolated and challenged with two different strains of influenza A viruses, PR8 and NIBRG-14. After 24 hours, we monitored infection by influenza A viruses using the IFA assay. We observed that the replication of the 2 influenza A strains was profoundly decreased in fibroblasts transgenic for Mx1 (Figure 4). Next, we monitored the viral growth curve by real time RT-PCR (Figure 5). When challenged with PR8 virus at an MOI of 1, we observed substantially lowered titers of PR8 virus in the transgenic versus the non-transgenic cells (Figure 5A). When the viral titers peaked at 15 hours post infection, there were approximately 10-fold less viral copies in the transgenic cells than in the non-transgenic cells. Following the peak at 15 h, viral titers began to decline, and this process occurred more rapidly in the Mx1 transgenic cells than in the non-transgenic controls. At 20 and 25 h post-infection, there was a 101.5 to 102-fold difference in the number of viral copies between the transgenic cells and the non-transgenic cells (Figure 5A, B). Although cells infected at an MOI of 0.01 produced overall lower viral titers than at an MOI of 1, they exhibited similar viral replication profiles and reduced viral copies in transgenic as compared to control cells. Similar differences in the kinetics of infection were observed between transgenic and non-transgenic cells when the NIBRG-14 influenza strain was used (Figure 5C, D).Figure 4


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)

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

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

Fig5: 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.
Mentions: Next we asked whether high expression levels of Mx1 rendered transgenic cells resistant to influenza A infection. To test this proposition, ear fibroblasts from transgenic pigs as well as non-transgenic controls were isolated and challenged with two different strains of influenza A viruses, PR8 and NIBRG-14. After 24 hours, we monitored infection by influenza A viruses using the IFA assay. We observed that the replication of the 2 influenza A strains was profoundly decreased in fibroblasts transgenic for Mx1 (Figure 4). Next, we monitored the viral growth curve by real time RT-PCR (Figure 5). When challenged with PR8 virus at an MOI of 1, we observed substantially lowered titers of PR8 virus in the transgenic versus the non-transgenic cells (Figure 5A). When the viral titers peaked at 15 hours post infection, there were approximately 10-fold less viral copies in the transgenic cells than in the non-transgenic cells. Following the peak at 15 h, viral titers began to decline, and this process occurred more rapidly in the Mx1 transgenic cells than in the non-transgenic controls. At 20 and 25 h post-infection, there was a 101.5 to 102-fold difference in the number of viral copies between the transgenic cells and the non-transgenic cells (Figure 5A, B). Although cells infected at an MOI of 0.01 produced overall lower viral titers than at an MOI of 1, they exhibited similar viral replication profiles and reduced viral copies in transgenic as compared to control cells. Similar differences in the kinetics of infection were observed between transgenic and non-transgenic cells when the NIBRG-14 influenza strain was used (Figure 5C, D).Figure 4

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