Production of α-1,3-galactosyltransferase (αGT)-deficient pigs is a fundamental task to overcome hyperacute rejection in pig-to-human xenotransplantation. However, the procedure to produce such pigs requires a great deal of cost, time, and labor. Heterozygote αGT knockout (KO) pigs should be bred at least for 2 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 fetal fibroblasts were transfected with αGT-targeting vector and treated with neomycin. Eighty-seven colonies were screened by PCR, and 2 among them were identified as homologous recombinants with monoallelic disruption of αGT (targeting efficiency = 2.3%). One of the two colonies was chosen and used for subsequent NT. In total, 559 nuclear transfer embryos reconstructed with αGT gene-targeted fetal fibroblasts were transferred to 5 surrogate gilts. Three gilts became pregnant and developed to term with 3 live piglets delivered. Being analyzed by PCR, all piglets born were found to be αGT gene targeted. Fibroblasts were isolated from ear skin biopsies of a 10-day-old αGT gene-targeted miniature pig. To increase the number of mitotic recombination that occurs spontaneously during cell proliferation, fibroblasts were cultured for 6 to 8 passages. Then, these cells were treated with IB4-conjugated Dynabeads; IB4 lectin binds to galactose-α-1,3-galactose (αGal), the antigen formed by αGT, and is responsible for hyperacute rejection (HAR). Using a magnetic column, fibroblasts bound to IB4 lectin were removed. Remaining cells lacking αGal were cultured further until colony formation. A total of 22 fibroblast colonies were screened by PCR, and 15 among them were confirmed as loss of heterozygosity (LOH) with biallelic disruption of αGT by mitotic recombination (LOH efficiency = 68%). One of these clonal cultures of αGT-deficient fibroblasts was chosen and used for subsequent NT. In total, 729 nuclear transfer embryos reconstructed with αGT-deficient neonatal ear skin fibroblasts were transferred to 4 surrogate gilts. Two gilts became pregnant, and 3 live piglets were produced. Based on PCR, both αGT alleles were targeted in all piglets born. Only 8 months were required from the production of heterozygote to homozygote KO in contrast to approximately 2 years consumed for conventional breeding of heterozygotes. The present study demonstrates that the time required for the production of αGT-deficient miniature pigs could be reduced significantly by neonatal skin biopsies and subsequent selection of mitotic recombinants. Such a procedure may be beneficial for the production of other homozygote KO animals, especially in species that require substantial periods of gestation such as pigs.
Production of -1,3-galactosyltransferase null pigs by means of nuclear transfer with fibroblasts bearing loss of heterozygosity mutations
