The enzyme α1,3-galactosyltransferase (α1,3GT or GCTA1) synthesizes α1,3-galactose (α1,3Gal) epitopes (Galα1,3Galβ1,4GlcNAc-R), which are the major xenoantigens causing hyperacute rejection in pig-to-human xenotransplantation. Complete removal of α1,3Gal from pig organs is the critical step toward the success of xenotransplantation. We reported earlier the targeted disruption of one allele of the α1,3GT gene in cloned pigs. A selection procedure based on a bacterial toxin was used to select for cells in which the second allele of the gene was knocked out. Sequencing analysis demonstrated that knockout of the second allele of the α1,3GT gene was caused by a T-to-G single point mutation at the second base of exon 9, which resulted in inactivation of the α1,3GT protein. Four healthy α1,3GT double-knockout female piglets were produced by three consecutive rounds of cloning. The piglets carrying a point mutation in the α1,3GT gene hold significant value, as they would allow production of α1,3Gal-deficient pigs free of antibiotic-resistance genes and thus have the potential to make a safer product for human use.The enzyme α1,3-galactosyltransferase (α1,3GT or GGTA1) synthesizes α1,3Gal epitopes (Galα1,3Galβ1,4GlcNAc-R) on the cell surface of almost all mammals with the exception of humans, apes, and Old World monkeys (1). α1,3Gal epitopes are the major xenoantigens causing hyperacute rejection (HAR) in pig-to-human xenotransplantation (2-4). Many reports have also indicated that α1,3Gal epitopes are involved in acute vascular rejection (AVR) of xenografts (4-6). Piglets with α1,3GT heterozygous knockout have been cloned Copyright © 2003 by our group (7) and another team (8) in the last year. To produce homozygous α1,3GT knockout piglets by natural breeding, assuming both male and female heterozygous knockout pigs are available at the same time and are fertile, is feasible but takes up to 12 months. However, by using a second-round knockout and cloning strategy, we could save up to 6 months and all cloned piglets would be α1,3GT double knockout (DKO). We have selected and enriched for α1,3GT DKO cells by using a bacterial toxin, toxin A from Clostridium difficile, which binds with high affinity to α1,3Gal epitopes and produces a cytotoxic effect on cells that are α1,3Gal-positive (9). Toxin A uses α1,3Gal epitopes as a cell surface receptor and causes "rounding" and lifting of the α1,3Gal-positive cells from the surface of the growth vessel (10, 11).Heterozygous α1,3GT knockout fetal fibroblasts, 657A-I11 1-6 cells, were isolated from a day-32 pregnancy as described in (7). To avoid using a second antibiotic-resistance gene as a selection marker, we constructed an ATG (start codon)-targeting α1,3GT knockout vector, pPL680 (12), which also contains a neo gene, to knock out the second allele of the α1,3GT gene. 657A-I11 1-6 cells were transfected by electroporation with pPL680 and selected for the α1,3Gal-negative phenotype with purified C. difficile toxin A (13). One colony (680B1) was isolated and expanded af...

Since the first report of live mammals produced by nuclear transfer from a cultured differentiated cell population in 1995 (ref. 1), successful development has been obtained in sheep, cattle, mice and goats using a variety of somatic cell types as nuclear donors. The methodology used for embryo reconstruction in each of these species is essentially similar: diploid donor nuclei have been transplanted into enucleated MII oocytes that are activated on, or after transfer. In sheep and goat pre-activated oocytes have also proved successful as cytoplast recipients. The reconstructed embryos are then cultured and selected embryos transferred to surrogate recipients for development to term. In pigs, nuclear transfer has been significantly less successful; a single piglet was reported after transfer of a blastomere nucleus from a four-cell embryo to an enucleated oocyte; however, no live offspring were obtained in studies using somatic cells such as diploid or mitotic fetal fibroblasts as nuclear donors. The development of embryos reconstructed by nuclear transfer is dependent upon a range of factors. Here we investigate some of these factors and report the successful production of cloned piglets from a cultured adult somatic cell population using a new nuclear transfer procedure.

Galactose-alpha1,3-galactose (alpha1,3Gal) is the major xenoantigen causing hyperacute rejection in pig-to-human xenotransplantation. Disruption of the gene encoding pig alpha1,3-galactosyltransferase (alpha1,3GT) by homologous recombination is a means to completely remove the alpha1,3Gal epitopes from xenografts. Here we report the disruption of one allele of the pig alpha1,3GT gene in both male and female porcine primary fetal fibroblasts. Targeting was confirmed in 17 colonies by Southern blot analysis, and 7 of them were used for nuclear transfer. Using cells from one colony, we produced six cloned female piglets, of which five were of normal weight and apparently healthy. Southern blot analysis confirmed that these five piglets contain one disrupted pig alpha1,3GT allele.

Recombinant adenoviruses containing the canine factor IX (FIX) cDNA were directly introduced in the hind leg muscle of mice. We show that (i) in nude mice, high expression (1-5 ,ug/ml in plasma) of FIX protein can be detected for >300 days; (ii) in contrast, expression of FIX protein was transient (7-10 days) in normal mice; (iii) CD8+ lymphocytes could be detected within 3 days in the infected muscle tissue; (iv) use of i32-microglobulin and immunoglobulin M heavy chain "knockout" mice showed that lack of sustained expression of FIX protein is due to cell-mediated and humoral immune responses; (v) normal mice, once infected with recombinant adenovirus, could not be reinfected efficiently for at least 30 days due to neutralizing viral antibodies; and, finally, (vi) using immunosuppressive drugs, some normal mice can be tolerized to produce and secrete FLIX protein for >5 months. We conclude that currently available adenoviral vectors have serious limitations for use for longterm gene therapy.Gene therapy holds a great promise for the treatment of many genetic diseases. Retroviral vectors have been extensively used to deliver genes into a wide variety of cell types but require ex vivo approaches because of their inability to infect postmitotic cells. In recent years much attention has been focused on a vector system that can deliver genes with high efficiency to a wide spectrum of nondividing cells in vivo. In particular, recombinant adenoviruses have been demonstrated to be very useful (1-5). Unfortunately, several studies with different gene products have revealed that following infection in a wide variety of target tissues, only transient expression is observed (6-12). Since the cells infected with the recombinant adenoviruses are rapidly eliminated, it is likely that the host immune system plays a major role in preventing sustained expression of the foreign genes. We have therefore undertaken a characterization of the specific immune responses and antigens involved in the lack of sustained expression by adenoviral factor IX (FIX) mediated gene therapy. in pXCJL1 were cotransfected with the pJM17 plasmid (14) into 293 cells (15), respectively, and recombinant adenoviral plaques were isolated and further purified by two rounds of plaque assays as described (16). The adenoviral vectors were propagated in 293 cells and purified by double CsCl banding as described (16). The purified virus was dialyzed against phosphate-buffered saline (PBS) and stored in aliquots with 15% glycerol at -80°C. MATERIALS AND METHODSAnimal Procedures. Adult Swiss Webster mice and nude (nu/nu) athymic mice were purchased from Harlan-SpragueDawley. 132-Microglobulin knockout, 132m(-/-), mice (17) and IgM heavy chain (,t chain) knockout, ,uMT/,uMT mice (18) were kindly provided by Leonard Shultz (The Jackson Laboratory). About 1 x 109 plaque-forming units (pfu) of purified AdMCdF9 or AdMCLacZ virus was diluted into 100 ,ul of PBS and injected into muscles of both hind legs of anesthetized adult mice at 6-10 weeks of age (5-10 ,ul ...

We transplanted kidneys from alpha1,3-galactosyltransferase knockout (GalT-KO) pigs into six baboons using two different immunosuppressive regimens, but most of the baboons died from severe acute humoral xenograft rejection. Circulating induced antibodies to non-Gal antigens were markedly elevated at rejection, which mediated strong complement-dependent cytotoxicity against GalT-KO porcine target cells. These data suggest that antibodies to non-Gal antigens will present an additional barrier to transplantation of organs from GalT-KO pigs to humans.

Two barriers prevent adenovirus-based vectors from having wide application. One is the difficulty of making new adenoviruses, and the second is the strong immunological reaction to viral proteins. Here we describe uses of Cre-lox recombination to overcome these problems. First, we demonstrate a simple method for constructing E1-substituted adenoviruses. Second, we demonstrate a method to construct adenovirus vectors carrying recombinant genes in place of all of the viral genes, so-called gutless adenovirus vectors. The pivotal feature in each method is the use of a negatively selected adenovirus named psi5. We engineered a cis-acting selection into psi5 by flanking its packaging site with loxP sites. When psi5 was grown in cells making a high level of Cre recombinase, the packaging site was deleted by recombination and the yield of psi5 was reduced to 5% of the wild-type level. To make a new E1-substituted virus, we used psi5 as a donor virus and recombined it with a shuttle vector via a loxP site. The resulting recombinant virus has a single loxP site next to the packaging site and therefore outgrows psi5 in the presence of Cre recombinase. To make a gutless virus, we used psi5 as a helper virus. The only viral sequences included in the gutless vector are those needed in cis for its replication and packaging. We found that a loxP site next to the packaging site of the gutless virus was necessary to neutralize homologous recombination between psi5 and the gutless viruses within their packaging domains.

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 ...

We have explored the use of primary myoblasts as a somatic tissue for gene therapy of acquired and inherited diseases where systemic delivery of a gene product may have therapeutic effects. Mouse primary myoblasts were infected with replication-defective retroviruses expressing canine factor IX cDNA under the control of a mouse muscle creatine kinase enhancer and human cytomegalovirus promoter. The infected myoblasts were injected into the hindlegs of recipient mice and levels of secreted factor IX protein were monitored in the plasma. We report sustained expression of factor EX protein for over 6 months without any apparent adverse effect on the recipient mice.