Efficient generation of a biallelic knockout in pigs using zinc-finger nucleases

<jats:p>Zinc-finger nucleases (ZFNs) are powerful tools for producing gene knockouts (KOs) with high efficiency. Whereas 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 (<jats:italic>GGTA1</jats:italic>) gene. Primary porcine fibroblasts were treated with ZFNs designed against the region coding for the catalytic core of<jats:italic>GGTA1</jats:italic>, resulting in biallelic knockout of ∼1% of ZFN-treated cells. A galactose (Gal) epitope 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<jats:italic>GGTA1</jats:italic>-KO phenotype was confirmed by a complement lysis assay that demonstrated protection of<jats:italic>GGTA1</jats:italic>-KO fibroblasts relative to wild-type cells. Cells from<jats:italic>GGTA1</jats:italic>-KO fetuses and pooled, transfected cells were used to produce live offspring via SCNT. This study reports the production of cloned pigs carrying a biallelic ZFN-induced knockout of an endogenous gene. These findings open a unique avenue toward the creation of gene KO pigs, which could benefit both agriculture and biomedicine.</jats:p>