The ??-1,3-Galactosyltransferase Knockout Mouse

Tearle, R; Tange, M. J.; Zannettino, Z. L.; Katerelos, Marina; Shinkel, Trixie A.; Denderen, Bryce J. W. van; Lonie, Andrew; Lyons, Ian; Nottle, Mark B.; Cox, Thomas C.; Becker, Carl G.; Peura, Anita; Wigley, Peter L.; Crawford, Robert J.; Robins, Allan J.; Pearse, Martin J.; d’Apice, Anthony J.F.

doi:10.1097/00007890-199601150-00004

Cited by 284 publications

(81 citation statements)

References 21 publications

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“…Southern blot and sequencing analysis of DNA samples from all five piglets confirmed the targeted disruption of the first allele of the α1,3GT gene and the T-to-G point mutation in the second base of exon 9 in the second allele of the α1,3GT gene. It has been reported that α1,3GT DKO mice developed cataracts at 4 to 6 weeks of age (17). Physical examination of the four piglets at 7 weeks of age did not reveal any abnormalities or cataracts.…”

mentioning

confidence: 72%

Production of α1,3-Galactosyltransferase-Deficient Pigs

Phelps¹,

Koike²,

Vaught³

et al. 2003

Science

1,019

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The enzyme α1,3-galactosyltransferase (α1,3GT or GCTA1) synthesizes α1,3-galactose (α1,3Gal) epitopes (Galα1,3Galβ1,4GlcNAc-R), which are the major xenoantigens causing hyperacute rejection in pig-to-human xenotransplantation. Complete removal of α1,3Gal from pig organs is the critical step toward the success of xenotransplantation. We reported earlier the targeted disruption of one allele of the α1,3GT 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 α1,3GT gene was caused by a T-to-G single point mutation at the second base of exon 9, which resulted in inactivation of the α1,3GT protein. Four healthy α1,3GT double-knockout female piglets were produced by three consecutive rounds of cloning. The piglets carrying a point mutation in the α1,3GT gene hold significant value, as they would allow production of α1,3Gal-deficient pigs free of antibiotic-resistance genes and thus have the potential to make a safer product for human use.The enzyme α1,3-galactosyltransferase (α1,3GT or GGTA1) synthesizes α1,3Gal epitopes (Galα1,3Galβ1,4GlcNAc-R) on the cell surface of almost all mammals with the exception of humans, apes, and Old World monkeys (1). α1,3Gal epitopes are the major xenoantigens causing hyperacute rejection (HAR) in pig-to-human xenotransplantation (2-4). Many reports have also indicated that α1,3Gal epitopes are involved in acute vascular rejection (AVR) of xenografts (4-6). Piglets with α1,3GT heterozygous knockout have been cloned Copyright © 2003 by our group (7) and another team (8) in the last year. To produce homozygous α1,3GT knockout piglets by natural breeding, assuming both male and female heterozygous knockout pigs are available at the same time and are fertile, is feasible but takes up to 12 months. However, by using a second-round knockout and cloning strategy, we could save up to 6 months and all cloned piglets would be α1,3GT double knockout (DKO). We have selected and enriched for α1,3GT DKO cells by using a bacterial toxin, toxin A from Clostridium difficile, which binds with high affinity to α1,3Gal epitopes and produces a cytotoxic effect on cells that are α1,3Gal-positive (9). Toxin A uses α1,3Gal epitopes as a cell surface receptor and causes "rounding" and lifting of the α1,3Gal-positive cells from the surface of the growth vessel (10, 11).Heterozygous α1,3GT knockout fetal fibroblasts, 657A-I11 1-6 cells, were isolated from a day-32 pregnancy as described in (7). To avoid using a second antibiotic-resistance gene as a selection marker, we constructed an ATG (start codon)-targeting α1,3GT knockout vector, pPL680 (12), which also contains a neo gene, to knock out the second allele of the α1,3GT gene. 657A-I11 1-6 cells were transfected by electroporation with pPL680 and selected for the α1,3Gal-negative phenotype with purified C. difficile toxin A (13). One colony (680B1) was isolated and expanded af...

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mentioning

confidence: 72%

Production of α1,3-Galactosyltransferase-Deficient Pigs

Phelps¹,

Koike²,

Vaught³

et al. 2003

Science

1,019

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“…That loss of the GGTA1 gene can be deleterious has become evident by the production of GGTA1 KO mice and pigs for xenotransplantation research (21,22,44). Animals with GGTA1 KO have both subtle and conspicuous health abnormalities.…”

Section: Discussionmentioning

confidence: 99%

Functionally important glycosyltransferase gain and loss during catarrhine primate emergence

Koike

Uddin

Wildman

et al. 2007

Proc. Natl. Acad. Sci. U.S.A.

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A glycosyltransferase, ␣1,3galactosyltransferase, catalyzes the terminal step in biosynthesis of Gal␣1,3Gal␤1-4GlcNAc-R (␣Gal), an oligosaccharide cell surface epitope. This epitope or antigenically similar epitopes are widely distributed among the different forms of life. Although abundant in most mammals, ␣Gal is not normally found in catarrhine primates (Old World monkeys and apes, including humans), all of which produce anti-␣Gal antibodies from infancy onward. Natural selection favoring enhanced resistance to ␣Gal-positive pathogens has been the primary reason offered to account for the loss of ␣Gal in catarrhines. Here, we question the primacy of this immune defense hypothesis with results that elucidate the evolutionary history of GGTA1 gene and pseudogene loci. One such locus, GGTA1P, a processed (intronless) pseudogene (PPG), is present in platyrrhines, i.e., New World monkeys, and catarrhines but not in prosimians. PPG arose in an early ancestor of anthropoids (catarrhines and platyrrhines), and GGTA1 itself became an unprocessed pseudogene in the late catarrhine stem lineage. Strong purifying selection, denoted by low nonsynonymous substitutions per nonsynonymous site/synonymous substitutions per synonymous site values, preserved GGTA1 in noncatarrhine mammals, indicating that the functional gene product is subjected to considerable physiological constraint. Thus, we propose that a pattern of alternative and/or more beneficial glycosyltransferase activity had to first evolve in the stem catarrhines before GGTA1 inactivation could occur. Enhanced defense against ␣Gal-positive pathogens could then have accelerated the replacement of ␣Gal-positive catarrhines by ␣Gal-negative catarrhines. However, we emphasize that positively selected regulatory changes in sugar chain metabolism might well have contributed in a major way to catarrhine origins. adaptive evolution ͉ glycobiology ͉ pseudogene

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“…Additional manipulation(s) may be a prerequisite to enhance the expression of A and B antigens. These may include the knockout of the ␣1,3-galactosyltransferase gene (44) or the introduction of the ␣1,2-fucosyltransferase gene under strong promoter (45) to either abolish or lessen the expression of the ␣1,3-galactosyltransferase. These mice with different ABO phenotypes in the same genetic background may clarify the functionality of the ABO system in the future.…”

Section: Musculus Domesticusmentioning

confidence: 99%

Murine Equivalent of the Human Histo-blood Group ABO Gene Is acis-AB Gene and Encodes a Glycosyltransferase with Both A and B Transferase Activity

Yamamoto

Lin

Kominato

et al. 2001

Journal of Biological Chemistry

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We have cloned murine genomic and complementary DNA that is equivalent to the human ABO gene. The murine gene consists of at least six coding exons and spans at least 11 kilobase pairs. Exon-intron boundaries are similar to those of the human gene. Unlike human A and B genes that encode two distinct glycosyltransferases with different donor nucleotide-sugar specificities, the murine gene is a cis-AB gene that encodes an enzyme with both A and B transferase activities, and this cis-AB gene prevails in the mouse population. Cloning of the murine AB gene may be helpful in establishing a mouse model system to assess the functionality of the ABO genes in the future.Histo-blood group A/B antigens are clinically important antigens in blood transfusion and organ transplantation. These antigens are oligosaccharide antigens whose immunodominant structures are defined as GalNAc ␣133 (Fuc ␣132) Gal-and Gal ␣133 (Fuc ␣132) Gal-for A and B antigen, respectively. Functional alleles at the ABO locus encode enzymes that catalyze the final step of synthesis. A alleles encode for A transferase, which transfers the GalNAc residues from the UDPGalNAc nucleotide-sugar to the galactose residue of the acceptor H substrates defined by Fuc ␣132 Gal-. B alleles encode for B transferase that transfers the galactose residue from UDP-galactose to the same H substrates. O alleles are nonfunctional, null alleles. During the past decade, we have been studying the molecular genetic basis of the histo-blood group ABO system (1). From a human gastric carcinoma cell line cDNA library, we were able to clone human A transferase cDNA (2) based on the partial amino acid sequence of the soluble form of A transferase purified from human lung (3). Using cross-hybridization with A transferase cDNA probes, we then cloned B transferase cDNA and nonfunctional O allelic cDNA from cDNA libraries made with RNA from colon adenocarcinoma cell lines that exhibited different ABO phenotypes (4). Possible allele-specific mutations were identified. Four amino acid substitutions were discovered between A and B transferases. O alleles were more homologous to A alleles than to B alleles. A single base deletion was found near the N terminus of the coding sequence in most of the O alleles, which caused the codon frame to shift. This resulted in a truncated protein without glycosyltransferase activity. In addition to the three major alleles (A

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The ??-1,3-Galactosyltransferase Knockout Mouse

Cited by 284 publications

References 21 publications

Production of α1,3-Galactosyltransferase-Deficient Pigs

Production of α1,3-Galactosyltransferase-Deficient Pigs

Functionally important glycosyltransferase gain and loss during catarrhine primate emergence

Murine Equivalent of the Human Histo-blood Group ABO Gene Is acis-AB Gene and Encodes a Glycosyltransferase with Both A and B Transferase Activity

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