Production of GGTA1 Targeted Pigs Using TALEN-Mediated Genome Editing Technology

Pigs are suggested to be an ideal species for xenotransplantation as they share similar anatomical and physiological characteristics with humans. Application of pigs for xenotransplantation had been hampered because of difficulty in generating genetically engineered (GE) pigs. Recent developments in genome editing technology using meganucleases demonstrate an efficient way of producing GE pigs. In this study, we examined the effectiveness of transcription activator-like effector nuclease (TALEN) system  in generating specific mutations on the pig genome. Our target gene was the α1,3-galactosyltransferase (GGTA1) as it is the main cause of hyperacute rejection after xenotransplantation. Specific TALENs were designed to induce a double-strand break on the exon 9 of porcine GGTA1 gene. Human decay-accelerating factor (hDAF) gene, which can produce a complement inhibitor to protect cells from complement attack after xenotransplantation, was also integrated into genome simultaneously. Plasmids coding for the TALEN pair and hDAF gene were transfected into porcine fetal fibroblasts by electroporation to disrupt porcine GGTA1 gene and express hDAF. Then the transfected cells were sorted using a biotin-labeled IB4 lectin, attached to magnetic beads, to obtain GGTA1 deficient cells. As a result, we established GGTA1 knock-out cell lines with biallelic modification (61/289, 21%) and GGTA1 knock-out cell lines expressing hDAF (6/46, 13%), respectively. When these cells were used for somatic cell nuclear transfer, we successfully obtained 6 live GGTA1 knockout pigs expressing hDAF from 3 surrogates. Our results demonstrate that TALEN-mediated genome editing is efficient and can be successfully used to generate GE pigs.