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Abstract Mad cow disease is caused by the prion protein-protease-resistant form (PrPsc), which is an isoform of protease-sensitive isoform of prion protein(PrPc), the aggregation of PrPsc in the central nervous system can result in the transmissible spongiform encephalopathy(TSE). By cutting down PrPc's expression to reach the purpose of decreasing its conversion into PrPsc is an effective way to inhibit TSE. In this article, we used the technology of RNA interference(RNAi) to cut down PrPc's expression, three candidate short hairpin RNAs(shRNAs) (prnp 1, prnp 2 and prnp 3) targeting the coding sequence of bovine prnp gene and a negative control (prnp -NC) were designed, then they were ligated into the vector of pGenesil-1 respectively, and were transfected into bovine fibroblast cells. By the means of Real-time PCR and Western blot, we could examine their suppression efficiency, at the same time, we selected cells in cell culture medium with G418 under a concentration of 700 μg/mL. The results showed that the interference effects of the three shRNA vectors were 51.2%, 85.7% and 74.6% respectively, the negative control's efficiency was 2.05%, so we obtained a more efficacious shRNA, and the positive cell clones were also got. This study provides a way for producing therapeutic transgenic animals by somatic cell nuclear transfer.
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Received: 25 November 2009
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Corresponding Authors:
Yong ZHANG
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