Inhibition Of Gene Expression Of Prnp In Bovine Fetal Fibroblast Cells By RNAi Technology

The mad cow disease is the Bovine spongiform Eneephalopathy's(BSE) popular name. It is an infectious, chronic, lethal central nervous system recession disease which is caused by the prion protein– PrPsc. PrPsc is the isoform of normal prion protein PrPc, they are coded by prnp gene. Along with the aggregation of PrPsc in the central nervous system, it can result to the transmissible bovine spongiform encephalopathy. By cutting down PrPc's expression to reach the purpose of decreasing its conversion into PrPsc is an effective way to inhibit BSE. RNAi is a highly efficient and specific gene knockdown technology, it can specially degrad it's homology sequence's mRNA in the cells, inhibit endogenous gene expression, then achieve the effect similar to gene knockout. So we employed RNA interference technology to knockdown prnp expression, it will contribute to deep research of this gene.SiRNA expression vector can continuously generate siRNA in the cells, make a long time gene silencing. And because it carries the antibiotic resistance gene, we can conduct drug screening to get the stably transfected cells, this is suitable for long time research. In this article, we used the technology of RNAi to cut down PrPc's expression, three candidate shRNAs targeting the CDS of bovine prnp gene and a negative control were designed, after reverse transcription, restriction enzyme cutting site and loop structure introduction, they were ligated into the vector of pGenesil-1 respectively, then we successfully constructed 4 RNAi-vector targeted to prnp, they were termed Prnp1, Prnp2, Prnp3 and Prnp–NC respectively. We transfected the four vectors into bovine fibroblast cells separately, 2 days and 3 days after transfection, we collected the transient transfected cells with fluorescence by flow cytometry(FCM)in sterile channel, then examined their suppression efficiency by the means of Real-time PCR and Western blot, the results showed that the interference effects of the three shRNA vectors were 51.2%,85.7% and 74.6%, the negative control's efficiency is 2.05%, so we obtained a more efficacious shRNA―Prnp2. After that, we used 700μg/mL G418 selected the fibroblast cells transfected with Prnp2, after 15 days, the positive cell clones were obatained.In this study, we combined the technology of FCM selected the transient transfected positive cells, then detected their interference efficiency, this can avoid the side effect of lower transfection efficiency. Except that, we obtained positive cell clones by the means of G418 selection, it provides a way for later study.