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Originally published in Science Express on 19 December 2002
Science 17 January 2003:
Vol. 299. no. 5605, pp. 411 - 414
DOI: 10.1126/science.1078942
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Reports

Production of alpha 1,3-Galactosyltransferase-Deficient Pigs

Carol J. Phelps,1 Chihiro Koike,34 Todd D. Vaught,1 Jeremy Boone,1 Kevin D. Wells,1 Shu-Hung Chen,1 Suyapa Ball,1 Susan M. Specht,34 Irina A. Polejaeva,1 Jeff A. Monahan,1 Pete M. Jobst,1 Sugandha B. Sharma,34 Ashley E. Lamborn,1 Amy S. Garst,1 Marilyn Moore,2 Anthony J. Demetris,35 William A. Rudert,36 Rita Bottino,36 Suzanne Bertera,36 Massimo Trucco,36 Thomas E. Starzl,34 Yifan Dai,1* David L. Ayares1*

The enzyme alpha 1,3-galactosyltransferase (alpha 1,3GT or GGTA1) synthesizes alpha 1,3-galactose (alpha 1,3Gal) epitopes (Galalpha 1,3Galbeta 1,4GlcNAc-R), which are the major xenoantigens causing hyperacute rejection in pig-to-human xenotransplantation. Complete removal of alpha 1,3Gal from pig organs is the critical step toward the success of xenotransplantation. We reported earlier the targeted disruption of one allele of the alpha 1,3GT 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 alpha 1,3GT gene was caused by a T-to-G single point mutation at the second base of exon 9, which resulted in inactivation of the alpha 1,3GT protein. Four healthy alpha 1,3GT double-knockout female piglets were produced by three consecutive rounds of cloning. The piglets carrying a point mutation in the alpha 1,3GT gene hold significant value, as they would allow production of alpha 1,3Gal-deficient pigs free of antibiotic-resistance genes and thus have the potential to make a safer product for human use.

1 PPL Therapeutics Inc., 1700 Kraft Drive, Blacksburg, VA 24060, USA.
2 PPL Therapeutics Ltd., Roslin, Midlothian, EH25 9PP, UK.
3 Thomas E. Starzl Transplantation Institute,
4 Department of Surgery,
5 Department of Pathology, and
6 Department of Pediatrics (Division of Immunogenetics) of University of Pittsburgh Medical Center (UPMC) and Children's Hospital, Pittsburgh, PA 15213, USA.
*   To whom correspondence should be addressed. E-mail: ydai{at}ppl-therapeutics.com; dayares{at}ppl-therapeutics.com

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