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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

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.
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Fig1: 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.

Mentions: Genomic DNA was extracted from the ear tissues of newborn cloned pigs for PCR analysis. The following primers were used to amplify the Mx1 gene fragments: Mx1-1: 5’- CAAATGGAGTGCTGTGGTTG-3’, Mx1-2: 5’-GCAGTACACGATCTCCA-3’, Mx1-5: 5’- ACAGGAGCGACAATTTTAAGC-3’ and Mx1-7: 5’- CGCCTTCACAGATGTTTCAG-3’. The binding sites of these primers are shown in Figure 1B. The expected sizes of PCR products of the transgenic Mx1 and endogenous Mx1 genes differ due to the presence of introns in the endogenous Mx1. Total RNA was extracted from fibroblasts isolated from the ear tissues of newborn cloned piglets using the RNeasy Mini Kit (Qiagen, Valencia, CA, USA), subjected to reverse transcription (PrimeScript RT Master Mix, Takara, Dalian, China) and real time RT-PCR (SYBR Premix Ex Taq™, Takara, Dalian, China) to determine the expression levels of Mx1 mRNA. The expression values for the Mx1 mRNA were normalized to the expression values obtained for GAPDH. The following primers were used for real time RT-PCR: Mx1-q1: 5’-CACAGAACTGCCAAGTCCAA-3’, Mx1-q2: 5’-GCAGTACACGATCTGCTCCA-3’, GAPDH-q1: 5’-CAGCAATGCCTCCTGTACCA-3’ and GAPDH-q2: 5’-GATGCCGAAGTTGTCATGGA-3’. RNA samples from 3 newborn natural breeding Tibet miniature piglets were used as controls. Western blot were used to detect the expression of Mx1 protein in the transgenic pigs as described above using fibroblast lysates and organ lysates of transgenic pig and non-transgenic pig control.Figure 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)

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

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

Fig1: 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.
Mentions: Genomic DNA was extracted from the ear tissues of newborn cloned pigs for PCR analysis. The following primers were used to amplify the Mx1 gene fragments: Mx1-1: 5’- CAAATGGAGTGCTGTGGTTG-3’, Mx1-2: 5’-GCAGTACACGATCTCCA-3’, Mx1-5: 5’- ACAGGAGCGACAATTTTAAGC-3’ and Mx1-7: 5’- CGCCTTCACAGATGTTTCAG-3’. The binding sites of these primers are shown in Figure 1B. The expected sizes of PCR products of the transgenic Mx1 and endogenous Mx1 genes differ due to the presence of introns in the endogenous Mx1. Total RNA was extracted from fibroblasts isolated from the ear tissues of newborn cloned piglets using the RNeasy Mini Kit (Qiagen, Valencia, CA, USA), subjected to reverse transcription (PrimeScript RT Master Mix, Takara, Dalian, China) and real time RT-PCR (SYBR Premix Ex Taq™, Takara, Dalian, China) to determine the expression levels of Mx1 mRNA. The expression values for the Mx1 mRNA were normalized to the expression values obtained for GAPDH. The following primers were used for real time RT-PCR: Mx1-q1: 5’-CACAGAACTGCCAAGTCCAA-3’, Mx1-q2: 5’-GCAGTACACGATCTGCTCCA-3’, GAPDH-q1: 5’-CAGCAATGCCTCCTGTACCA-3’ and GAPDH-q2: 5’-GATGCCGAAGTTGTCATGGA-3’. RNA samples from 3 newborn natural breeding Tibet miniature piglets were used as controls. Western blot were used to detect the expression of Mx1 protein in the transgenic pigs as described above using fibroblast lysates and organ lysates of transgenic pig and non-transgenic pig control.Figure 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