Establishment Of A Cell Line Expressing PrP/GFP Fusion Protein And Its Application In PrP Expression Inhibition Test By RNA Interference

In vivo transmission studies have clearly demonstrated the inverse correlation between the expression level of PrPc in host animal and the incubation time of prion disease. Cell lines highly expressing PrPc are considered to be ideal models for both research and diagnosis in vitro. We use the eukaryotic expression plasmid pCDNA3.1(-) to transfer PrP/GFP fusion cassette into the Neuro-2a cell line to generate a new cell line with increased yield of PrPc.PCR primers were designed according to the published sequence of mouse PrP gene and the GFP sequence of pEGFP plasmid. The amplified DNA fragments were inserted into the pGEM-T vector separately to generate two recombinant plasmids, namely, pGEM-mPrP and pGEM-GFP. Plasmids pGEM-mPrP, pGEM-GFP and pCDNA3.1(-) were then digested with EcoRI and BamHI, BamHI and HindIII, EcoRI and HindIII respectively. The recovered bands of PrP, GFP and pCDNA3.1(-) were ligated with T4 ligase to produce a fusion expression plasmid, pCDNA3.1(-)-mPG, which was then used to transfect the Neuro-2a cell under the selection pressure of G418 antibiotics. The cells were subject to 30 passages under the selection and checked for the expression of PrP/GFP fusion protein by fluorescence and western blotting. The evidence of the fusion gene in the genome of cell line was confirmed by PCR. The final cell line was named as mPGN2a.RNA interference (RNAi), which specifically inhibits protein expression, is a new approach to the study of protein function.To find if small interference RNA could inhibit the PrP expression, the specific...