earticle

영어

In the pig industry, foot and mouth disease (FMD) and porcine reproductive and respiratory syndrome (PRRS) cause lots of social and economic losses annually. Many resourses have been used to prevent these viruses, however, vaccinations have obvious limitation on viral diseases. It is difficult to prevent the diseases, because viruses constantly change their genetic information naturally. Therefore, genetic modification of the host has been studied to overcome the limitations of vaccine. In this study, disease resistance was introduced with two genetic modification methods. First the PRRSV viral receptor (CD163) was edited with the CRISPR/Cas9 technique. Porcine fetal fibroblasts (pFF) with the CRISPR/Cas9 vector were single cell cultured to make unique cell lines. As results, CD163 sequences of edited cells showed difference with control cells. Second, shRNAs that target 3D gene (encoding FMDV RNA polymerase) and ORF7 (encoding PRRSV nucleocapsid protein) were introduced into pFF. Those techniques above were combined into single cell line by introducing shRNAs into CD163-edited cells. Vectors expressing the 3D gene and ORF7 gene were lipofected into the cells to confirm shRNA activity in vitro. The cells containg shRNAs showed significantly lower level of transcript than the control cells (about 9-fold in 3D and 160- fold in ORF7). The multi-resistant cells also showed decreasing in transcript level of viral genes (more than 4.8-fold in 3D and 3.8-fold in ORF7). With all these results, we obtained the multi-resistant cell line against FMD and PRRS, and established in vitro shRNA verification system successfully.