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.

Viable pigs exhibiting robust and ubiquitous expression of pCTLA4-Ig were produced on both a WT and GTKO background. Expression of pCTLA4-Ig resulted in acute susceptibility to opportunistic pathogens due at least in part to a significantly compromised humoral immune status. As this molecule is known to have immunosuppressive activity, high levels of pCTLA4-Ig expression in the blood, as well as defective development related to exposure to pCTLA4-Ig in utero, may contribute to this reduced immune status. Prophylactic treatment with antibiotics may promote survival of disease-free transgenic pigs to a size optimal for organ procurement for transplantation. Additional genetic modifications and/or tightly regulated expression of pCTLA4Ig may reduce the impact of this transgene on the humoral immune system.

The stable long-term expression of anti-PERV siRNAs was shown to be effective in knocking down PERV expression in cells. However, the very low (sometimes undetectable), and variable levels of expression of PERV in normal pigs make it difficult to obtain suitable control animals for comparison, to assess knockdown of PERV in vivo. This was demonstrated by the observation that even cloned non-transgenic littermates, express levels of PERV as low as that of some of their siRNA transgenic littermates. Further analysis is required to conclusively quantitate in vivo effects in the shRNA transgenic pigs.