Alpha-1,3-galactosyltransferase-deficient miniature pigs produced by serial cloning using neonatal skin fibroblasts with loss of heterozygosity.

Abstract Production of alpha-1,3-galactosyltransferase (αGT)-deficient pigs is essential to overcome xenograft rejection in pig-to-human xenotransplantation. However, the procedure to produce such pigs requires a great deal of cost, time, and labor. Heterozygous αGT knockout pigs should be bred at least for two generations to ultimately produce homozygote progenies. Here, we show that αGT-deficient miniature pigs could be produced in much reduced time using mitotic recombination in neonatal ear skin fibroblasts. Miniature pig fibroblasts were transfected with αGT gene-targeting vector. Resulting gene-targeted fibroblasts were used for nuclear transfer (NT), and three live αGT gene-targeted piglets were produced from cloned embryos. Fibroblasts isolated from ear skin biopsies of these piglets were cultured for 6-8 passages to induce loss of heterozygosity (LOH) and treated with biotin-conjugated IB4 that binds to galactose-α-1,3-galactose, an epitope produced by αGT. Using magnetic activated cell sorting, cells with monoallelic disruption of αGT were removed. Remaining cells with LOH carrying biallelic disruption of αGT were confirmed by PCR and used for NT. Three homozygous αGT knockout live piglets were produced from the transfer of cloned embryos to surrogates. The present study demonstrates that the time required for the production of αGT-deficient miniature pigs could be reduced significantly by postnatal skin biopsies and subsequent selection of mitotic recombinants. Such procedure may be beneficial for the production of homozygote knockout animals, especially in species, such as pigs, that requires substantial length of time for breeding.