Establishment of prion deficient cells in cattle

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Animal cloning techniques have enabled gene disruption in several species. Here, we report the first successful disruption of the prion gene in cattle. METHODS : The prion gene of the Japanese Black cow (JBC) was used to construct PrP-5. 1 and PrP-5. 2, the targeting vectors for the bovine prion gene, and these constructs were introduced into the fetal fibroblast cell line JBC906 by the lipofection method. Ten polymerase chain reaction (PCR)-positive colonies were obtained from 833 G418-resistant colonies, and Southern blot analysis revealed successful homologous recombination at the prion locus in these colonies. CONCLUSIONS : Heterozygous knockout of the prion gene was performed in JBC, and production of a homozygous prion knockout JBC by a second round of targeting 906htGT is currently in progress.