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.
Highlights
The genetic modification of swine can have a multitude of agricultural and medical applications
The transfer of multiple foreign genes into pigs is necessary for generating models of various human diseases, such as Alzheimer’s disease and Parkinson’s disease, which are related to multiple genes or factors [7,8]
The enhanced green fluorescent proteins (EGFP) and enhanced cyan fluorescent protein (ECFP) cDNAs were inserted into pBStdTomato-2A and pBS-ZsYellow1-2A generating pBS-tdTomato-2A-EGFP and pBS-ZsYellow1-2A-ECFP, respectively, to construct a vector in which the expressions of the four fluorescent genes were driven by two independent CAG promoters
Summary
The genetic modification of swine can have a multitude of agricultural and medical applications. Specific genetic modifications in swine may afford organs for pigto-human xenotransplantation, produce recombinant products for biomedical or nutraceutical use, as well as generate models of human diseases for medical research and drug development [3,5,6]. In agriculture, it might be necessary to produce pigs with several favorable traits (e.g., low fat/muscle ratio, high disease resistance, and enriched with special nutrients), requiring multiple corresponding genes to be transferred into the pigs. The transfer of multiple foreign genes into pigs is necessary for generating models of various human diseases, such as Alzheimer’s disease and Parkinson’s disease, which are related to multiple genes or factors [7,8]. Organs from multitransgenic pigs expressing two or three complement genes involved in immunological rejection have exhibited prolonged xenograft survival [9,10,11,12,13]
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