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...

Here we describe a procedure for cloning pigs by the use of in vitro culture systems. Four healthy male piglets from two litters were born following nuclear transfer of cultured somatic cells and subsequent embryo transfer. The initiation of five additional pregnancies demonstrates the reproducibility of this procedure. Its important features include extended in vitro culture of fetal cells preceding nuclear transfer, as well as in vitro maturation and activation of oocytes and in vitro embryo culture. The cell culture and nuclear transfer techniques described here should allow the use of genetic modification procedures to produce tissues and organs from cloned pigs with reduced immunogenicity for use in xenotransplantation.

Central to the success of large animal cloning is the production of healthy animals that can provide products for human health, food, and other animal agriculture applications. We report development of cloned cattle derived from 34 genetically unique, nonembryonic cell lines using nuclear transfer performed between 1 January 1998 and 29 February 2000. Nearly 25% (535/2170) of the recipients receiving reconstructed embryos initiated pregnancy. Overall, 19.8% (106/535) of the initiated pregnancies resulted in live births, while 77% (82/106) of these cattle clones remain healthy and productive today. Although a wide variation in birth weight of clone calves was observed, their growth rates, reproductive performance, and lactation characteristics are similar to that found in noncloned dairy cattle. Our data represent the most comprehensive information on cattle derived from nuclear transfer procedures and indicate that this emerging reproductive technology offers unique opportunities to meet critical needs in both human health care and agriculture.

The pregnancy initiation and maintenance rates of nuclear transfer embryos produced from several bovine cell types were measured to determine which cell types produced healthy calves and had growth characteristics that would allow for genetic manipulation. Considerable variability between cell types from one animal and the same cell type from different animals was observed. In general, cultured fetal cells performed better with respect to pregnancy initiation and calving than adult cells with the exception of cumulous cells, which produced the highest overall pregnancy and calving rates. The cell type that combined relatively high pregnancy initiation and calving rates with growth characteristics that allowed for extended proliferation in culture were fetal genital ridge (GR) cells. Cultured GR cells used in nuclear transfer and embryo transfer initiated pregnancies in 40% of recipient heifers (197), and of all recipients that received nuclear transfer embryos, 9% produced live calves. Cultured GR cells doubled as many as 85 times overall and up to 75 times after dilution to single-cell culture. A comparison between transfected and nontransfected cells showed that transfected cells had lower pregnancy initiation (22% versus 32%) and calving (3.4% versus 8.9%) rates.

The ability to modify animal genomes rapidly at a specific locus would be valuable both for research purposes and in the development of animals suitable for xenotransplantation. In a proof-of-concept study, we used a unique, homology-dependent strand transferase protein called drosophila recombination-associated protein (DRAP) and DNA oligonucleotides to modify the porcine gene encoding alpha 1,3 galactosyl transferase (GGTA1). This gene is responsible for generating xenotransplantation antigens resulting in hyperacute rejection. Pronuclear injection of DRAP and mutant oligonucleotides yielded piglets with heritable, modified alleles of GGTA1 in a direct, rapid and efficient manner. Cells derived from these piglets had markedly reduced alpha 1,3 galactosyl sugar epitopes. The simplicity of this method should permit rapid sequential or simultaneous modification of the various genes encoding or producing antigens that impose limits on xenotransplantation as they are discovered.

This is the first report of intrauterine Mycobacterium paratuberculosis transmission from a bovine embryo recipient to the resultant calf. The recipient was ELISA and fecal culture test-negative before embryo placement, but became ELISA-positive during the pregnancy. Johne’s disease was confirmed at necropsy subsequent to Caesarean section. Her embryo transfer calf was diagnosed with Johne’s disease two years later. Strict research biosecurity protocols under which the embryo transfer calf was raised eliminated routes of post-natal exposure to M. paratuberculosis. The recipient cow had been purchased from a herd at risk for M. paratuberculosis infection for which no Johne’s disease surveillance was performed.To minimize the likelihood of purchasing an infected recipient cow or obtaining M. paratuberculosis- contaminated colostrum and thus jeopardizing valuable embryos, recipient and colostrum donor purchases should be made from a source herd with no or low, whole- herd Johne’s disease test prevalence. The risk of purchasing an animal with M. paratuberculosis infection can be better assessed with whole-herd infection prevalence data than with test results for a single animal only.