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Targeted disruption of the α1,3-galactosyltransferase gene in cloned pigs


Galactose-α1,3-galactose (α1,3Gal) is the major xenoantigen causing hyperacute rejection in pig-to-human xenotransplantation. Disruption of the gene encoding pig α1,3-galactosyltransferase (α1,3GT) by homologous recombination is a means to completely remove the α1,3Gal epitopes from xenografts. Here we report the disruption of one allele of the pig α1,3GT 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 α1,3GT allele.

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Figure 1: Disruption of the porcine α1,3GT gene by gene targeting.
Figure 2: Southern blot analysis of α1,3GT gene knockout fetuses and piglets.
Figure 3


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We thank B. Gragg, W. Lucero, T. Akers, H. Bishop, and J. McPherson for technical contributions to embryo transfer and animal husbandry; A. Garst and J. Hencke for help in nuclear transfer; C. Koike and T. E. Starzl at the University of Pittsburgh for providing us with porcine α1,3GT gene genomic sequence; M. Moore, A. Kind, and A. Schnieke for valuable contributions in discussions; and staff at the Virginia Maryland Regional Veterinary College for prenatal care of some of the nuclear transfer recipients and physical examination of piglets. This research was funded in part by a grant from the National Institute of Standards and Technology (NIST) Advanced Technology Program (ATP).

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Correspondence to Yifan Dai.

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Dai, Y., Vaught, T., Boone, J. et al. Targeted disruption of the α1,3-galactosyltransferase gene in cloned pigs. Nat Biotechnol 20, 251–255 (2002).

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