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Use of the 2A peptide for generation of multi-transgenic pigs through a single round of nuclear transfer.

Deng W, Yang D, Zhao B, Ouyang Z, Song J, Fan N, Liu Z, Zhao Y, Wu Q, Nashun B, Tang J, Wu Z, Gu W, Lai L - PLoS ONE (2011)

Bottom Line: The reconstructed embryos, which were obtained by somatic cell nuclear transfer and confirmed to express the four fluorescent proteins evenly, were transplanted into seven recipient gilts.Eleven piglets were delivered by two gilts, and seven of them co-expressed the four fluorescent proteins at equivalently high levels in various tissues.The fluorescence intensities were directly observed at the nose, hoof and tongue using goggles.

View Article: PubMed Central - PubMed

Affiliation: Key Laboratory of Regenerative Biology, South China Institute for Stem Cell Biology and Regenerative Medicine, Chinese Academy of Sciences, Guangzhou, China.

ABSTRACT
Multiple genetic modifications in pigs can essentially benefit research on agriculture, human disease and xenotransplantation. Most multi-transgenic pigs have been produced by complex and time-consuming breeding programs using multiple single-transgenic pigs. This study explored the feasibility of producing multi-transgenic pigs using the viral 2A peptide in the light of previous research indicating that it can be utilized for multi-gene transfer in gene therapy and somatic cell reprogramming. A 2A peptide-based double-promoter expression vector that mediated the expression of four fluorescent proteins was constructed and transfected into primary porcine fetal fibroblasts. Cell colonies (54.3%) formed under G418 selection co-expressed the four fluorescent proteins at uniformly high levels. The reconstructed embryos, which were obtained by somatic cell nuclear transfer and confirmed to express the four fluorescent proteins evenly, were transplanted into seven recipient gilts. Eleven piglets were delivered by two gilts, and seven of them co-expressed the four fluorescent proteins at equivalently high levels in various tissues. The fluorescence intensities were directly observed at the nose, hoof and tongue using goggles. The results suggest that the strategy of combining the 2A peptide and double promoters efficiently mediates the co-expression of the four fluorescent proteins in pigs and is hence a promising methodology to generate multi-transgenic pigs by a single nuclear transfer.

Show MeSH
Genotype identification of multi-transgenic piglets.(A) Positions of the two primer pairs for genotype identification. Genomic DNA from Piglet 2, 3, 4, 6, 7, 9, 10 was extracted, whereas ZC (B) and TG (C) bicistronic cassettes in the genome were detected by PCR using appropriate primers. Lane 1, DNA marker; lane 2, positive control; lane 3, negative control; lane 4–10, genomic DNA from Piglet 2, 3, 4, 6, 7, 9 and 10.
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pone-0019986-g003: Genotype identification of multi-transgenic piglets.(A) Positions of the two primer pairs for genotype identification. Genomic DNA from Piglet 2, 3, 4, 6, 7, 9, 10 was extracted, whereas ZC (B) and TG (C) bicistronic cassettes in the genome were detected by PCR using appropriate primers. Lane 1, DNA marker; lane 2, positive control; lane 3, negative control; lane 4–10, genomic DNA from Piglet 2, 3, 4, 6, 7, 9 and 10.

Mentions: Genomic DNA extracted from the ear fibroblasts of Piglets 2, 3, 4, 6, 7, 9 and 10 was used for PCR to confirm the presence of the four fluorescent protein genes in the genome of the cloned piglets. As shown in Figure 3, these piglets contained the four fluorescent protein genes, confirming that they were multi-transgenic pigs.


Use of the 2A peptide for generation of multi-transgenic pigs through a single round of nuclear transfer.

Deng W, Yang D, Zhao B, Ouyang Z, Song J, Fan N, Liu Z, Zhao Y, Wu Q, Nashun B, Tang J, Wu Z, Gu W, Lai L - PLoS ONE (2011)

Genotype identification of multi-transgenic piglets.(A) Positions of the two primer pairs for genotype identification. Genomic DNA from Piglet 2, 3, 4, 6, 7, 9, 10 was extracted, whereas ZC (B) and TG (C) bicistronic cassettes in the genome were detected by PCR using appropriate primers. Lane 1, DNA marker; lane 2, positive control; lane 3, negative control; lane 4–10, genomic DNA from Piglet 2, 3, 4, 6, 7, 9 and 10.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3094386&req=5

pone-0019986-g003: Genotype identification of multi-transgenic piglets.(A) Positions of the two primer pairs for genotype identification. Genomic DNA from Piglet 2, 3, 4, 6, 7, 9, 10 was extracted, whereas ZC (B) and TG (C) bicistronic cassettes in the genome were detected by PCR using appropriate primers. Lane 1, DNA marker; lane 2, positive control; lane 3, negative control; lane 4–10, genomic DNA from Piglet 2, 3, 4, 6, 7, 9 and 10.
Mentions: Genomic DNA extracted from the ear fibroblasts of Piglets 2, 3, 4, 6, 7, 9 and 10 was used for PCR to confirm the presence of the four fluorescent protein genes in the genome of the cloned piglets. As shown in Figure 3, these piglets contained the four fluorescent protein genes, confirming that they were multi-transgenic pigs.

Bottom Line: The reconstructed embryos, which were obtained by somatic cell nuclear transfer and confirmed to express the four fluorescent proteins evenly, were transplanted into seven recipient gilts.Eleven piglets were delivered by two gilts, and seven of them co-expressed the four fluorescent proteins at equivalently high levels in various tissues.The fluorescence intensities were directly observed at the nose, hoof and tongue using goggles.

View Article: PubMed Central - PubMed

Affiliation: Key Laboratory of Regenerative Biology, South China Institute for Stem Cell Biology and Regenerative Medicine, Chinese Academy of Sciences, Guangzhou, China.

ABSTRACT
Multiple genetic modifications in pigs can essentially benefit research on agriculture, human disease and xenotransplantation. Most multi-transgenic pigs have been produced by complex and time-consuming breeding programs using multiple single-transgenic pigs. This study explored the feasibility of producing multi-transgenic pigs using the viral 2A peptide in the light of previous research indicating that it can be utilized for multi-gene transfer in gene therapy and somatic cell reprogramming. A 2A peptide-based double-promoter expression vector that mediated the expression of four fluorescent proteins was constructed and transfected into primary porcine fetal fibroblasts. Cell colonies (54.3%) formed under G418 selection co-expressed the four fluorescent proteins at uniformly high levels. The reconstructed embryos, which were obtained by somatic cell nuclear transfer and confirmed to express the four fluorescent proteins evenly, were transplanted into seven recipient gilts. Eleven piglets were delivered by two gilts, and seven of them co-expressed the four fluorescent proteins at equivalently high levels in various tissues. The fluorescence intensities were directly observed at the nose, hoof and tongue using goggles. The results suggest that the strategy of combining the 2A peptide and double promoters efficiently mediates the co-expression of the four fluorescent proteins in pigs and is hence a promising methodology to generate multi-transgenic pigs by a single nuclear transfer.

Show MeSH