title: Inhibition of porcine endogenous retrovirus in PK15 cell line by efficient multitargeting RNA interference.
abstract: To effectively suppress porcine endogenous retroviruses (PERV)s, RNAi technique was utilized. RNAi is the up-to-date skill for gene knockdown which simultaneously multitargets both gag and pol genes critical for replication of PERVs. Previously, two of the most effective siRNAs (gag2, pol2) were found to reduce the expression of PERVs. Concurrent treatment of these two siRNAs (gag2+pol2) showed knockdown efficiency of up to 88% compared to negative control. However, despite the high initial knockdown efficiency 48 h after transfection caused by siRNA, it may only be a transient effect of suppressing PERVs. The multitargeting vector was designed, containing both gag and pol genes and making use of POL II miR Expression Vector, which allowed for persistent and multiple targeting. This is the latest shRNA system technique expressing and targeting like miRNA. Through antibiotics resistance characteristics utilizing this vector, miRNA-transfected PK15 cells (gag2-pol2) were selected during 10 days. An 88.1% reduction in the level of mRNA expression was found. In addition, we performed RT-activity analysis and fluorescence in situ hybridization assay, and it demonstrated the highest knockdown efficiency in multitargeting (gag2+pol2) miRNA group. Therefore, according to the results above, gene knockdown system (siRNA and shRNA) through multitargeting strategy could effectively inhibit PERVs.
authors.affiliation: Department of Veterinary Medicine Virology Laboratory, College of Veterinary Medicine and Research Institute for Veterinary Science Seoul National University, Seoul, Korea.
authors.forename: Hee-Chun
authors.initials: HC
authors.lastname: Chung
authors.forename: Van-Giap
authors.initials: VG
authors.lastname: Nguyen
authors.forename: Hyoung-Joon
authors.initials: HJ
authors.lastname: Moon
authors.forename: Hye-Kwon
authors.initials: HK
authors.lastname: Kim
authors.forename: Seong-Jun
authors.initials: SJ
authors.lastname: Park
authors.forename: Jee-Hoon
authors.initials: JH
authors.lastname: Lee
authors.forename: Min-Gyung
authors.initials: MG
authors.lastname: Choi
authors.forename: A-Reum
authors.initials: AR
authors.lastname: Kim
authors.forename: Bong-Kyun
authors.initials: BK
authors.lastname: Park
mesh_terms: D000818:Animals; D002460:Cell Line; D018920:Coculture Techniques; D020077:Endogenous Retroviruses / Q000502:physiology*; D055785:Gene Knockdown Techniques; D015750:Genes, gag / Q000187:drug effects; D015751:Genes, pol / Q000187:drug effects; D057809:HEK293 Cells; D006801:Humans; D034622:RNA Interference; D013552:Swine
keywords: PK15 cells; RNA interference; inhibition; multitargeting; porcine endogenous retrovirus