Use of the 2A Peptide for Generation of Multi-Transgenic Pigs through a Single Round of Nuclear Transfer

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

Expression profile analysis of four fluorescent proteins in various tissues by RT-PCR.

<p>Total mRNA was extracted from various tissues of Piglet 7. The mRNA levels of the target genes (tdTomato, EGFP+ECFP and ZsYellow1) were determined by RT-PCR. The results are shown as mean ± S.D.</p

Development ability of nuclear transfer embryos derived from pZCpTG-PFFs and WT-PFFs.

<p>The embryo numbers are the total numbers counted from three independent experiments, and the percentage values in the third and fourth columns are shown as mean ± S.D.</p><p>*<i>p</i>>0.05 compared with WT-PFF; no significant difference.</p

Maps of the pTGZC (A) and pZCpTG (B) vectors.

<p>Maps of the pTGZC (A) and pZCpTG (B) vectors.</p

Genotype identification of multi-transgenic piglets.

<p>(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.</p

Co-expression of the four fluorescent proteins in the fibroblast isolated from multi-transgenic piglets.

<p>(A) Fibroblasts isolated from the ear tissue of a representative animal (Piglet 10) observed under a confocal microscope using appropriate filters. The scale bar represents 10 µm. (B) The fluorescence intensities of the four fluorescent proteins were measured. The results are shown as mean ± S.D.</p

Fluorescence in living multi-transgenic piglets.

<p>Florescence on the noses and hooves of multi-transgenic piglets was directly observed using goggles. (A) Bright light. (B) tdTomato fluorescence. (C) EGFP fluorescence. (D) ECFP fluorescence. A WT piglet was used as negative control.</p

Co-expression of the four fluorescent proteins in PFFs and blastocysts.

<p>(A) PFFs transfected with the linearized pZCpTG vector were observed under a confocal microscope using appropriate filters. The scale bar represents 10 µm. (B) Several reconstructed embryos derived from the pZCpTG PFFs were cultured until they reached the blastocyst stage. The blastocysts were observed under a fluorescence microscope using appropriate filters.</p