The PRNP gene encodes a cellular protein named prion, whose misfolded form has been implicated in a number of neuropathic diseases in mammals such as the Bovine Spongiform Encephalopathy (BSE) in cattle. BSE has brought devastating impact on the world economy and human health. Recently, several groups have performed the gene targeting strategy to disrupt the PRNP gene in bovine fibroblast cells and produce BSE-resistant cattle by somatic cell nuclear transfer (SCNT). However, the enrichment efficiency of the gene targeting vector was low. Here, we constructed a novel promoterless gene targeting vector to sequentially disrupt the PRNP gene in bovine fibroblast cells and generate gene targeted cattle by SCNT. The enrichment efficiency of the novel vector was 100% and 60%, respectively. After nuclear transfer, no significant difference was found in the rate of cleavage and blastocyst formation between the knockout and wild type cloned embryos. One PRNP⁺/⁻ calf was born with no obvious abnormal development by now. Fusion RT-PCR and real-time PCR showed one allele of the PRNP gene was functionally disrupted, and the mRNA expression reduced dramatically in the PRNP⁺/⁻ cattle. The reconstituted PRNP⁻/⁻ embryos showed double alleles disruption, and no difference in the rate of cleavage and blastocyst formation.
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