Targeted disruption of the alpha1,3-galactosyltransferase gene in cloned pigs.

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.

Efficient production of transgenic cloned calves using preimplantation screening.

The genetic manipulation of donor cells before nuclear transfer (NT) enables prior selection for transgene integration. However, selection for genetically modified cells using antibiotic drugs often results in mixed populations, resulting in a mixture of transgenic and nontransgenic donor cells for NT. In this study, we attempted to develop efficient strategies for the generation of human bile salt-stimulated lipase (BSSL) transgenic cows. Preimplantation screening by either biopsy or green fluorescent protein (GFP) expression was used to detect NT-derived BSSL transgenic embryos to ensure that the calf born would be transgenic. We compared the development rates of NT-derived embryos from G418- and GFP-selected donor cells. There were no significant differences (P < 0.001) in cleavage rate (67.2% vs. 60.0%) and blastocyst formation rate (44.9% vs. 41.2%). We also compared the pregnancy rates of the G418/biopsy and GFP preimplantation screened NT-derived blastocysts. The Day 40 pregnancy rate of the G418/biopsy group (40%) was lower than that of the GFP group (57%), but the calf birth rate of the G418/biopsy group (40%) was higher than that of the GFP group (21%). Healthy BSSL transgenic calves were born after both screening processes. This is the first report of biopsy-screened cloned transgenic animals. The results suggest that both selection methods are useful for detecting transgenic NT embryos without negatively affecting their development into viable transgenic offspring.

Production of alpha 1,3-galactosyltransferase-deficient pigs.

The enzyme alpha1,3-galactosyltransferase (alpha1,3GT or GGTA1) synthesizes alpha1,3-galactose (alpha1,3Gal) epitopes (Galalpha1,3Galbeta1,4GlcNAc-R), which are the major xenoantigens causing hyperacute rejection in pig-to-human xenotransplantation. Complete removal of alpha1,3Gal from pig organs is the critical step toward the success of xenotransplantation. We reported earlier the targeted disruption of one allele of the alpha1,3GT 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 alpha1,3GT gene was caused by a T-to-G single point mutation at the second base of exon 9, which resulted in inactivation of the alpha1,3GT protein. Four healthy alpha1,3GT double-knockout female piglets were produced by three consecutive rounds of cloning. The piglets carrying a point mutation in the alpha1,3GT gene hold significant value, as they would allow production of alpha1,3Gal-deficient pigs free of antibiotic-resistance genes and thus have the potential to make a safer product for human use.