Heterozygous disruption of the alpha1,3-galactosyltransferase gene in cattle

Animal cloning techniques have enabled gene disruption in several species. Here, we report the first successful disruption of the alpha1,3-galactosyltransferase (alpha1,3-GT) gene in cattle.

The alpha1,3-GT gene of the Japanese Black cow (JBC) was used to construct pGT-6, a targeting vector for the bovine alpha1,3-GT gene, and pGT-6 was introduced into the fetal fibroblast cell line JBC906 by the lipofection method. Four polymerase chain reaction (PCR)-positive colonies were obtained from 797 G418-resistant colonies, and Southern blot analysis revealed successful homologous recombination at the alpha1,3-GT locus in one of the four colonies. Nuclear transfer was performed, and the four embryos were transferred to a heifer.

To establish fetal fibroblasts that were heterozygously disrupted at the alpha1,3-GT locus, one of the fetuses was recovered at 5 weeks of pregnancy, and PCR and Southern blot analysis of the fetal fibroblasts established from it showed definite homologous recombination of the alpha1,3-GT gene.

Heterozygous knockout of the alpha1,3-GT gene was performed in JBC, and production of a homozygous alpha1,3-GT knockout JBC by a second round of targeting 906htGT is currently in progress. The technique described here can be applied to disruption of other genes in cattle.