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

Abstract

Prion diseases, which are called transmissible spongiform encephalopathies (TSE), 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 down-regulation efficacy was increased in siRNA dose-dependent manner. The greatest down-regulation 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.

Figures and Tables

Figure 1 Rreal-time RT-PCR analysis to determine the level of mouse prion mRNA (A) and RT-PCR analysis to determine the expression of bovine prion mRNA (B) in NbP and GbP cells after transfection with different concentrations of siRNA. The quantity of mouse prion mRNA was normalized to that of GAPDH. An increase in siRNA resulted in a gradual suppression of the mouse prion mRNA, while the expression of bovine prion mRNA remained unchanged.

Figure 2 Western blot analysis to evaluate the abundance of mouse prion protein with 3F10 and prion proteins including moPrP and boPrP with SAF70 in each cell line after transfection of 100 nM of siRNA. Densitometric evaluation showed downregulated moPrP and stable expressed boPrP in NbPsiRNA cells (A) or GbPsiRNA cells (B) compared with siRNA non-treated cells.

Figure 3 Downregulation of prion expression by different concentrations of siRNA1 or siRNA2. Cells were transfected with siRNA1 (A) or siRNA2 (B) and incubated for 48 h. The level of the expressed prion protein was determined by western blot (above) and evaluated by densitometry (below). Increasing concentrations of siRNA resulted in a gradual suppression of prion protein expression.

Figure 4 Time-dependent inhibition of prion expression by siRNA-mediated knockdown. Cells were transfected with 100 nM siRNA. The levels of prion expression were determined by western blot (above) and evaluated by densitometry (below). The downregulation by each siRNA was effective within 72 h of transfection by more than 80%.

Table 1 Oligonucleotide sequences of primers, probe and siRNAs used in this study