Production of α-1,3-galactosyltransferase null pigs by means of nuclear transfer with fibroblasts bearing loss of heterozygosity mutations

Abstract

Hyperacute rejection of porcine organs by old world primate recipients is mediated through preformed antibodies against galactosyl-␣-1,3-galactose (Gal␣-1,3-Gal) epitopes expressed on the pig cell surface. Previously, we generated inbred miniature swine with a null allele of the ␣-1,3-galactosyltransferase locus (GGTA1) by nuclear transfer (NT) with gene-targeted fibroblasts. To expedite the generation of GGTA1 null pigs, we selected spontaneous null mutant cells from fibroblast cultures of heterozygous animals for use in another round of NT. An unexpectedly high rate of spontaneous loss of GGTA1 function was observed, with the vast majority of null cells resulting from loss of the WT allele. Healthy piglets, hemizygous and homozygous for the genetargeted allele, were produced by NT by using fibroblasts that had undergone deletional and crossover͞gene conversion events, respectively. Aside from loss of Gal␣-1,3-Gal epitopes, there were no obvious phenotypic differences between these null piglets and WT piglets from the same inbred lines. In fact, congenital abnormalities observed in the heterozygous NT animals did not reappear in the serially produced null animals.A ntibodies against galactosyl-␣-1,3-galactose (Gal␣-1,3-Gal) residues on cell surface glycoproteins of pig cells mediate hyperacute rejection of porcine organs in primate model recipients and are the most immediate barrier to successful clinical xenotransplantation (1, 2). High levels of preformed ''natural'' antibodies against the Gal␣-1,3-Gal epitope are found in humans and old world primates, following evolutionary loss of the corresponding galactosyltransferase activity (encoded by GGTA1) (3). The presence of these antibodies, along with the high density of Gal␣-1,3-Gal residues on most pig cells (4), suggests that elimination of GGTA1 function would provide a practical means of overcoming both hyperacute rejection and subsequent acute or chronic tissue damage associated with antibody binding to this epitope.The lack of GGTA1 function in humans and old world primates, along with the viability of GGTA1 knockout mice produced with embryonic stem cell technology (5, 6), suggested that a knockout strategy might be biologically feasible in pigs. The cloning of sheep (7) and subsequently pigs (8-10) by nuclear transfer with somatic cells has made attempts to knockout the GGTA1 locus in pigs technically feasible.We have previously reported the generation of GGTA1 heterozygous inbred miniature swine using nuclear transfer with gene-targeted fibroblasts (11). Starting with heterozygous fibroblasts from such animals, we now report the isolation of GGTA1 null cells with spontaneous loss of the WT allele. The rate of loss of heterozygosity (LOH) was several orders of magnitude greater than typically expected, an observation that may be related to the inbred background of the heterozygous animals. LOH resulted in some cases from deletion of the WT allele and in others from either somatic crossing over or gene conversion. Similarly high rates of somatic recombi...

Expression of X‐linked genes in deceased neonates and surviving cloned female piglets

“…Pigs have been regarded as promising donors for xenotransplantation and attempts have been made to genetically modify the porcine genome through somatic cell NT in order to make the porcine organs immunologically more compatible to human Kolber-Simonds et al 2004;Lai et al 2002;Ramsoondar et al 2003). The production of pigs through NT has been particularly difficult, probably due to the inefficiencies of in vitro oocyte maturation and embryo culture, and the necessity for at least four good-quality embryos to establish a pregnancy.…”

Rituximab Treatment Prevents the Early Development of Proteinuria following Pig-to-Baboon Xeno-Kidney Transplantation

“…With the advent of a1,3-galactosyltransferase (GalT) knockout donor animals (GalTKO), [1][2][3][4][5] xenograft survival in our pig-to-baboon kidney transplant model was extended to an average of .50 days. 6 Unfortunately, despite the achievement of immunologic hyporesponsiveness to pig antigens through a tolerance-inducing strategy using "thymokidney" grafts, 6 most of the recipients developed severe post-transplant proteinuria.…”

The possibilities of practical application of transgenic mammalian species generated by somatic cell cloning in pharmacology, veterinary medicine and xenotransplantology

“…In contrast to α-1,3-GT-semideficient cloned pigs, complete removal of galactosyl-α-1,3-galactose epitopes in the organs of α-1,3-GT-deficient cloned piglets may be the critical step toward the success of xenotransplantation (Phelps et al 2003). Kolber-Simonds et al (2004) produced healthy, totally α-1,3-GT-deficient, inbred miniature piglets with two distinct genotypes for loss of heterozygosity (LOH) in the GGTA1 locus, using serial nuclear transfer with fibroblast cells (Fig. 1).…”

“…Such a strategy can overcome both hyperacute rejection and subsequent acute or chronic tissue damage of porcine grafts in preclinical tests of pig-to-primate or human xenotransplantology. The former and latter processes are associated with the binding of preformed anty-Gal antibodies to the galactosyl-α-1,3-galactose residues on the surface of endothelial cells (Phelps et al 2003, Kolber-Simonds et al 2004.…”