33 PRODUCTION OF TRANSGENIC-CLONED PIGS CARRYING HDAF, GnT-III AND HETEROZYGOUSLY DISRUPTED α-1,3-GALACTOSYLTRANSFERASE GENES
T. Fujimura A , Y. Takahagi A , H. Nagashima C , S. Miyagawa B , T. Shigehisa C and H. Murakami AA The Animal Engineering Research Institute, Tsukuba, Ibaraki, Japan
B Division of Organ Transplantation, Department of Regenerative Medicine, Osaka University Graduate School of Medicine, Osaka, 565-0871, Japan
C Laboratory of Developmental Engineering, Department of Life Science, School of Agriculture, Meiji University, Kowasaki, 214-8571, Japan. Email: t.fujimura@nipponham.co.jp
Reproduction, Fertility and Development 17(2) 166-166 https://doi.org/10.1071/RDv17n2Ab33
Submitted: 1 August 2004 Accepted: 1 October 2004 Published: 1 January 2005
Abstract
In pig-to-human xenotransplantation, transplants are rapidly rejected by binding of human natural antibodies to porcine xenoantigen, mostly Gal α-1-3Gal oligosaccharides, and subsequent complement attack. To overcome this rejection, we so far have produced transgenic pigs expressing both human CD55/DAF (decay-accelerating factor, a complement-regulatory protein) and GnT-III (N-acetylglucosaminyltransferase III, a sugar chain modifying enzyme). In the present study, we heterozygously disrupted the α-1,3-galactosyltransferase (GT) gene, which catalyses the biosynthesis of Gal α-1-3Gal epitopes, in the fetal fibroblast cells from the DAF/GnT-III transgenic pigs by homologous recombination, and successfully produced GT-knockout pigs by nuclear transfer. Fibroblast cells isolated from Day 30 fetuses of DAF/GnT-III transgenic pigs were transfected with a GT-targeting vector. The targeting event in drug-resistant colonies was confirmed by PCR analysis, and targeted cells were used as nuclear donors. The reconstructed embryos were electrically activated and transferred to estrus-synchronized recipient pigs. At pregnancy Day 27 of gestation, fetuses were collected and their fibroblast cells were isolated for secondary nuclear transfer. The genomic DNA of live-born piglets produced by the secondary nuclear transfer were analyzed for the presence of DAF and GnT-III genes as well as the heterozygous disruption of the GT gene. From a total of 5.5 × 107 cells transfected with the GT-targeting vector, 2,749 drug-resistant colonies were obtained. Eighteen colonies were judged positive for targeting events by PCR analysis. After transfer of 321 cloned embryos reconstructed with the knockout cells to three recipients, four knockout fetuses were obtained from one recipient. Transfer of 633 cloned embryos reconstructed with the knockout fibroblast cells from one knockout fetus to six recipients gave rise to two live knockout piglets. PCR analysis of genomic DNA confirmed that the cloned piglets carried both DAF and GnT-III transgenes as well as the heterozygously disrupted GT gene.
