Efficient Production Of α1,3-galactosyltransferase Gene-deficient Wuzhishan Miniature Pigs And Its Function Analysis

The production of the a1,3-galactosyltransferase (GGTA1) gene-deficient Wuzhishan miniature pigs which can effectively overcome Hyperacute Rejection(HAR), will establish the gene targeting platform for somatic cells and provie the valuable genetically modificated animal models for the further development of the xenotransplantation between the pig and human. Basis on the Wuzhishan miniature pigs, we successfully selected the GGAT1-/-cells by different strategies combined Magnetic cell sorting, and production of the GGAT1-/-Wuzhishan miniature pigs. Establishment of the GGAT1-/-ear fibroblast lines, absences of a1,3Gal antigens in the GGAT1-/-cells were comfirmed by flow cytometric analysis, and resistances of cells were comfirmed by Cell Lysis.Successfully selected1×104spontaneous loss of heterozygosity (LOH) cells from fibroblast cultures of heterozygous animals (GGAT1+/-) using a biotin-labeled IB4lectin attached to streptavidin-coated magnetic beads. After additional cultivation,29single cell colonies were isolated. The colonies were confirmed by PCR,15GGAT1-/-cell colonies were obtained. The high rate of spontaneous loss of GGTA1function was more than7.4×10-4. Successfully selected4×105cells from fibroblast cultures lines which transferred Transcription Activator Like Effectors Nuclease (TALEN) using a biotin-labeled IB4lectin attached to streptavidin-coated magnetic beads. After additional cultivation,27single cell colonies were isolated. The colonies were confirmed by PCR,17GGAT1-/-and1GGAT1+/-cell colonies were obtained. The high rate of TALEN targeting was more than4.4×10-2.A total of4674somatic nuclear transferred embryos reconstructed with the GGTA1-/-cells were transferred into18recipient pigs, seven of them were pregnant, and producing24liveborns. Absences of GGATl in the cloned pigs were confirmed by PCR and Southern Blotting,23liveborns were GGAT1knockout piglets.Successful establishment of the GGAT1-/-ear fibroblast lines, absences of α1,3Gal antigens in the GGAT1-/-cells were comfirmed by flow cytometric analysis, and revealed that al,3Gal antigens were not present in the cells of the cloned GGAT1-/-pigs. The resistance of cells were comfirmed by Cell Lysis, the death percentage of GGAT1-/-cells were10%, while the the wild-type cells (GGTA1+/+) were50%, we demonstrated that GGAT1-/-cells of the cloned pigs can effectively overcome the HAR.The production of the a1,3-galactosyltransferase(GGATl) gene-deficient Wuzhishan miniature pigs will provide the valuable genetically modificated animal models,and a typical paradigm and an important resource for the further development of genetically modified pigs for make organs tailored for transplantation to humans.