Mouse neuronal cells expressing exogenous bovine PRNP and simultaneous downregulation of endogenous mouse PRNP using siRNAs

摘要

Prion diseases, which are called transmissible spongiform encephalopathies (TSEs), comprise a group of fatal infectious neurodegenerative disorders. Investigation of prion strains and generation of species dependent TSE model are necessary to understand pathogenesis of the disease. To establish a BSE-specific in vitro cell culture model, N2a and GT1 mouse neuronal cell lines were generated to express the bovine prion protein by transfection of the bovine prion gene (Prnp). In addition, the endogenous mouse prion protein was suppressed in N2a, NbP, GT1 and GbP cell lines using the siRNA duplexes, siRNA1 and siRNA2 that target the N- and C-termini of murine Prnp, respectively. Both siRNA1 and siRNA2 effectively decreased murine prion protein levels by more than 80% and the downregulation efficacy was increased in siRNA dose-dependent manner. The greatest downregulation was observed 48 h after siRNA delivery. The moPrnp knockdown NbP and GbP cell lines and the Prnp-targeting siRNA technique established in the present study would be useful tools for dissecting the basic mechanisms of prion infection, especially for BSE.