Knockdown of the prion gene expression by RNA interference in bovine fibroblast cells

Abstract

PRNP is the gene encoding prion protein whose misfolded and b-sheet-rich isoform is the infectious agent of transmissible spongiform encephalopathy (TSE). TSE, also called prion diseases, cause fatal neurodegenerative and transmissible disorders in human and animals. Among these diseases, bovine spongiform encephalopathy (BSE) has tremendous impact on economy and human health in the world. In the present study, we hypothesize suppression of the PRNP gene expression could raise resistance to BSE in cattle by using vectorbased small interfering RNA (siRNA) expression systems. Therefore, the objective was to screen effective DNAencoding short hairpin RNAs (shRNAs) which could knockdown the PRNP gene expression in bovine fibroblast cells. Human U6 promoter was employed to drive shRNA transcription from the DNA vector, and seven shRNAs, that designed to target coding region and 3 0 untranslated region of the PRNP gene, were selected. Four out of seven shRNAs tested were found to be effective in inhibiting the PRNP gene expression, and the most significant suppression level was as much as 62.9% evidenced by real-time RT-PCR. Furthermore, the protein abundance was obviously reduced compared to the control. Overall, the present study demonstrated that vectorbased siRNA expression systems is an efficient approach to knockdown the PRNP gene expression in bovine fibroblast cells and thereby provide donor cells for somatic cell nuclear cloning to produce cattle that is resistant to prion related diseases.

RNA Interference in Pigs: Comparison of RNAi Test Systems and Expression Vectors

“…RNAi reporter systems such as the Dual-Luciferase assay are commonly used to identify functional RNAi constructs [28,34,35]. In our hands, 12 of 16 RNAi sequences identified in silico led to significant signal reduction with this assay (see Tables 1, 2), a higher proportion than previously reported [33].…”

Neurotoxic Effect of the Complex of the Ovine Prion Protein (OvPrPC) and RNA on the Cultured Rat Cortical Neurons

“…Moreover, as a small molecule, RNA can activates aptamers and bind to functional pockets of proteins, and even be used to discriminate between conformational isoforms and normal forms of proteins [15,34,35].The prion protein complexed to N2a cellular RNAs via its N-terminal domain forming aggregates, which is toxic to murine neuroblastoma cells. PrP interacts with RNA with high affinity (nanomolar range) and this interaction leads to an insoluble aggregated protein [15].…”

Induction and Mechanism of Apoptosis by Hydroxycamptothecin in Human Tenon's Capsule Fibroblasts

“…Total protein content was determined using BCA assay as described previously, 23 and equal amounts of samples were separated using 12% SDS-PAGE. Samples were transferred to polyvinylidene difluoride membranes with a Bio-Rad transfer unit at 20 V for 1 hour at room temperature.…”