Generation of GGTA1 Biallelic Knockout Yucatan Miniature Pigs Via ZFNs and Somatic Cell Nuclear Transfer

Genetically modified pigs have been considered valuable models of human disease and donors for xenotransplantation. Here, we used Zinc finger nucleases (ZFNs) to knock out the Yucatan miniature pig α-1,3-galactosyltransferase (GGTA1) gene, which generates Gal epitopes that trigger hyperacute immune rejection in pig-to-human transplantation. ZFNs were designed to cleave a region of the GGTA1 gene. Biallelic GTKO cell lines were established from single cell colonies of ear fibroblasts derived from Yucatan miniature pigs following transfection by electroporation. Two cell lines were selected as donor cell line for somatic cell nuclear transfer (SCNT) for the generation of GTKO pigs. The reconstructed GTKO embryos were subsequently transferred into two recipient gilts, of which one became pregnant. We obtained four live piglets and one stillborn. Genotyping of all cloned individuals was performed. The Gal expression in the fibroblasts of all piglets was analyzed by fluorescence activated cell sorting (FACS) and western blotting. Sequencing analyses of the target site confirmed the homozygous GGTA1-null mutation in all fetuses and piglets, consistent with the genotype of the donor cells. Furthermore, FACS and western blotting analyses demonstrated that Gal epitopes were completely absent from the fibroblasts of all GTKO piglets.