Multi-resistance strategy for viral diseases and short hairpin RNA verification method in pigs

• Main Authors: Jong-nam Oh, Kwang-hwan Choi, Chang-kyu Lee
• Format: Article
• Language: English
• Published: Asian-Australasian Association of Animal Production Societies 2018-04-01
• Series: Asian-Australasian Journal of Animal Sciences
• Subjects: Pig; Viral Diseases; Foot and Mouth Disease (FMD); Porcine Reproductive and Respiratory Syndrome (PRRS); CRISPR-Cas9; RNAi
• Online Access: http://www.ajas.info/upload/pdf/ajas-31-4-489.pdf

Objective Foot and mouth disease (FMD) and porcine reproductive and respiratory syndrome (PRRS) are major diseases that interrupt porcine production. Because they are viral diseases, vaccinations are of only limited effectiveness in preventing outbreaks. To establish an alternative multi-resistant strategy against FMD virus (FMDV) and PRRS virus (PRRSV), the present study introduced two genetic modification techniques to porcine cells. Methods First, cluster of differentiation 163 (CD163), the PRRSV viral receptor, was edited with the clustered regularly interspaced short palindromic repeats-CRISPR-associated protein 9 technique. The CD163 gene sequences of edited cells and control cells differed. Second, short hairpin RNA (shRNAs) were integrated into the cells. The shRNAs, targeting the 3D gene of FMDV and the open reading frame 7 (ORF7) gene of PRRSV, were transferred into fibroblasts. We also developed an in vitro shRNA verification method with a target gene expression vector. Results shRNA activity was confirmed in vitro with vectors that expressed the 3D and ORF7 genes in the cells. Cells containing shRNAs showed lower transcript levels than cells with only the expression vectors. The shRNAs were integrated into CD163-edited cells to combine the two techniques, and the viral genes were suppressed in these cells. Conclusion We established a multi-resistant strategy against viral diseases and an in vitro shRNA verification method.