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DOI: https://doi.org/10.5713/ajas.17.0749    [Accepted] Published online December 19, 2017.
Multi-resistance strategy for viral diseases and in vitro shRNA verification method in pigs
Jong-nam Oh1, Kwang-hwan Choi1, C. K Lee1,2,*
1Department of Agricultural Biotechnology, Animal Biotechnology Major, Research Institute of Agriculture and Life Sciences, Seoul National University, Seoul, Korea
2Designed Animal and Transplantation Research Institute (DATRI), Institute of Green Bio Science and Technology, Seoul National University, Kangwon-do, Korea
Correspondence:  C. K Lee, Tel: 82+2-880-4805, Fax: 82+2-873-2271, Email: leeck@snu.ac.kr
Received: 13 October 2017   • Revised: 21 November 2017   • Accepted: 24 November 2017
Abstract
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, CD163, the PRRSV viral receptor, was edited with the CRISPR-Cas9 technique. The CD163 gene sequences of edited cells and control cells differed. Second, shRNAs were integrated into the cells. The shRNAs, targeting the 3D gene of FMDV and the 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.
Keywords: Pig; Viral diseases; Foot and mouth disease (FMD); porcine reproductive and respiratory syndrome (PR; CRISPR-Cas9; RNAi
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