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Zinc-finger nuclease mediated knockout of the porcine α1,3-galactosyltransferase gene

Janet Hauschild¹, Bjoern Petersen¹, Yolanda Santiago², Anna-Lisa Queisser¹, Joseph Carnwath¹, Andrea Lucas-Hahn¹, Lei Zhang², Xiangdong Meng², Philip Gregory², Reinhard Schwinzer³, Gregory Cost², Heiner Niemann¹

¹Institute of Farm Animal Genetics, Friedrich-Loeffler-Institut, Neustadt a. Rbge., Germany; ²Sangamo BioSciences, Inc., Richmond, CA, United States; ³Hannover Medical School, Transplantation Laboratory Hannover, Hannover, Germany

Zinc-finger nucleases (ZFNs) are powerful tools for producing gene knockouts (KOs) with high efficiency. While ZFN-mediated gene disruption has been demonstrated in laboratory animals such as mice, rats, and fruit flies, ZFNs have not been used to disrupt an endogenous gene in any large domestic species. Here we used ZFNs to induce a biallelic knockout of the porcine α1,3-galactosyltransferase (GGTA1) gene. Primary porcine fibroblasts were treated with ZFNs designed against the region coding for the catalytic core of GGTA1, resulting in biallelic knockout of ~1% of ZFN-treated cells. A galactose epitope (Gal)-counter-selected population of these cells was used in somatic cell nuclear transfer (SCNT). Of the resulting six fetuses, all completely lacked Gal epitopes and were phenotypically indistinguishable from the starting donor cell population, illustrating that ZFN-mediated genetic modification did not interfere with the cloning process. Neither off-target cleavage events nor integration of the ZFN-coding plasmid was detected. The GGTA1-KO phenotype was confirmed by a complement lysis assay that demonstrated protection of GGTA1-KO fibroblasts relative to wild-type cells. Cells from GGTA1-KO fetuses as well as pooled, transfected cells were used to produce live offspring via SCNT. In total, eight living GGTA1 -/- piglets were born. This study is the first to report the production of cloned pigs carrying a biallelic ZFN-induced knockout of an endogenous gene. These findings open a new avenue towards the creation of gene KO-pigs which could benefit both agriculture and biomedicine.