The presence of galactose alpha-1,3-galactose residues on the surface of pig cells is a major obstacle to successful xenotransplantation. Here, we report the production of four live pigs in which one allele of the alpha-1,3-galactosyltransferase locus has been knocked out. These pigs were produced by nuclear transfer technology; clonal fetal fibroblast cell lines were used as nuclear donors for embryos reconstructed with enucleated pig oocytes.

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...

Mammalian oocytes are arrested at the G2-M phase transition of the first meiotic division. In vitro, fully grown oocytes liberated from their follicles spontaneously reinitiate meiosis I, characterized by germinal vesicle breakdown (GVBD), chromatin condensation, spindle assembly, emission of the first polar body and progression to metaphase of the second meiotic division (MII), at which stage they undergo a second arrest until fertilization. After spermatozoa penetrate the oocyte, the second polar body extrudes, male and female pronuclei form and syngamy occurs to start early embryo development. Nuclear changes during oocyte maturation and fertilization are co-ordinated with movements of genetic material and organelles, and with biochemical changes in the cytoplasm to ensure normal embryo development. The normality of early embryogenesis is directly related to the ordered expression of these developmental programmes (Van Blerkom, 1991).Of the numerous cytoplasmic changes that occur, the positioning of mitochondria may be involved in concentrating ATP or calcium to specific regions in oocytes or fertilized eggs to support normal developmental processes. Thus, the distribution of active mitochondria may be indicative of the energy or ion requirement of various key events during oocyte maturation, fertilization and early embryo development. In mice, the perinuclear accumulation of mitochondria between GVBD and metaphase I (MI) (Van Blerkom and Runner, 1984; Van Blerkom, 1991), and the polarized distribution of mitochondria to one half of the oocyte containing the MII spindle (Calarco, 1995) were observed and were regarded as one aspect of the developmental programme of cytoplasmic maturation. Previous observations also revealed that translocation of mitochondria is co-ordinated

Meat products are generally low in omega-3 (n-3) fatty acids, which are beneficial to human health. We describe the generation of cloned pigs that express a humanized Caenorhabditis elegans gene, fat-1, encoding an n-3 fatty acid desaturase. The hfat-1 transgenic pigs produce high levels of n-3 fatty acids from n-6 analogs, and their tissues have a significantly reduced ratio of n-6/n-3 fatty acids (P < 0.001).The health benefits of long chain n-3 fatty acids, found mainly in fish oils, are well recognized. Meat products normally contain small amounts of n-3 fatty acids and large amounts of n-6 fatty acids 1 . Diets with a high ratio of n-6/n-3 fatty acids may contribute to the prevalence of many diseases, such as coronary artery disease, cancer, diabetes, arthritis and depression 2 . The high n-6/n-3 ratio in meat products is largely due to the extensive use of grains rich in n-6 fatty acids but deficient in n-3 fatty acids as animal feed. In addition, livestock cannot convert n-6 fatty acids into n-3 fatty acids because they lack an n-3 fatty acid desaturase gene, such as the fat-1 gene found in the roundworm C. elegans 3 . Earlier work in transgenic mice carrying the fat-1 gene has suggested the feasibility of creating fat-1 transgenic livestock capable of producing n-3 fatty acids from the corresponding n-6 fatty acids 4 . Here we report the cloning of fat-1 transgenic pigs that produce high levels of n-3 fatty acids in their tissues and organs. COMPETING INTERESTS STATEMENTThe authors declare competing financial interests (see the Nature Biotechnology website for details).Reprints and permissions information is available online at http://npg.nature.com/reprintsandpermissions/ NIH Public Access An hfat-1 expression vector, pCAGGS-hfat-1, which contains a humanized fat-1 cDNA (with modification of codon usage) driven by the cytomegalovirus enhancer and chicken β-actin promoter, has been described previously 4 . A pgk-neo expression cassette as a selection marker was inserted into pCAGGS-hfat-1 to generate pST103, which was transfected into earlypassage male primary porcine fetal fibroblast cells, pCFF4-3 5 , by e1ectroporation; the transfected cells were selected with 250 µg/ml G418. The G418-resistant colonies were pooled. Gas chromatographic analysis showed that pCFF4-3/pST103 cells contained higher amounts of n-3 fatty acids and lower amounts of n-6 fatty acids compared with the nontransfected pCFF4-3 cells, indicating that the hfat-1 protein was functional in the primary porcine cells. The PCFF4-3/pST103 cells were used to clone hfat-1 transgenic pigs by nuclear transfer as described previously 6 . A total of 1,633 reconstructed embryos were transferred into 14 gilts that exhibited a natural estrus. Twelve early pregnancies were established, and five of them went to term. Twelve (ten alive and two dead) male piglets were born by either caesarean section or natural delivery. PCR analysis of DNA samples from the tails of ten live piglets showed that six piglets (nos. 1, 3-5, 8 and 9) were positive ...