Gender non-specific efficacy of ZFN mediated gene targeting in pigs

Abstract

Zinc-finger nuclease (ZFN) mediated gene disruption is significantly more efficient than conventional targeting as homologous recombination (HR) and has been successfully applied in several species, including fruit fly, rats, human and the domestic pig (Bibikova et al. 2002; Urnov et al. 2005; Geurts et al. 2009; Hauschild et al. 2011). Biallelic gene knockouts (KOs) can be achieved within one step (Urnov et al. 2005). ZFNs induce a double-strand break (DSB) which is repaired by error prone non homologous end joining (NHEJ) DNA repair creating mutations which can lead to a gene KO. Recently, we have reported the biallelic KO of the porcine a1,3-galactosyltransferase (GGTA1, Gal) gene in female porcine fetal fibroblasts that were successfully used in somatic cell nuclear transfer (SCNT) and resulted in the birth of healthy piglets with a homozygous KO of GGTA1 (Hauschild et al. 2011). The GGTA1 gene encodes the Galepitopes on the porcine cell surface which primarily are responsible for the hyperacute rejection (HAR) after pig-to-human xenotransplantation. Organs of Gal-negative pigs transplanted to baboons have shown significantly improved organ survival (up to 236 days) (Mohiuddin et al. 2012). Initially, somatic cloning was only successful with female donor cells (Cibelli et al. 1998; Kato et al. 1998; Wakayama et al. 1998; Wilmut et al. 1997) and the question was raised whether or not male donor cells were equally efficient in somatic cloning as their female counterparts. The first male cloned offspring were produced from adult tale tip cells from mice (Wakayama and Yanagimachi 1999). Subsequently, it was shown that female and male donor cells were equally efficient in producing embryonic stem cells from cloned mouse blastocysts, and other factors such as donor cell type and genotype are more important with regard to cloning efficiency (Wakayama et al. 2005). Here, we demonstrate that male primary fibroblasts are compatible with a similar targeting and cloning efficiency as their female counterparts. This will facilitate routine production of GGTA1 pigs. Production of male ZFN-mediated GGTA1 KO cells was accomplished as described before (Hauschild et al. 2011). Biallelic GGTA1-KO rate (0.8 %) was comparable to the rate obtained in female porcine cells (1 %). SCNT using male Gal-negative cells resulted in five live GGTA1 piglets and five stillJ. Hauschild-Quintern B. Petersen (&) A.-L. Queisser A. Lucas-Hahn H. Niemann (&) Institute of Farm Animal Genetics, Friedrich-LoefflerInstitut, Hoeltystrasse 10, Mariensee, 31535 Neustadt a. Rbge., Germany e-mail: bjoern.petersen@fli.bund.de