title: Early lethality of shRNA-transgenic pigs due to saturation of microRNA pathways.
abstract: RNA interference (RNAi) is considered as a potential modality for clinical treatment and anti-virus animal breeding. Here, we investigate the feasibility of inhibiting classical swine fever virus (CSFV) replication by short hairpin RNA (shRNA) in vitro and in vivo. We generate four different shRNA-positive clonal cells and two types of shRNA-transgenic pigs. CSFV could be effectively inhibited in shRNA-positive clonal cells and tail tip fibroblasts of shRNA-transgenic pigs. Unexpectedly, an early lethality due to shRNA is observed in these shRNA-transgenic pigs. With further research on shRNA-positive clonal cells and transgenic pigs, we report a great induction of interferon (IFN)-responsive genes in shRNA-positive clonal cells, altered levels of endogenous microRNAs (miRNA), and their processing enzymes in shRNA-positive cells. What is more, abnormal expressions of miRNAs and their processing enzymes are also observed in the livers of shRNA-transgenic pigs, indicating saturation of miRNA/shRNA pathways induced by shRNA. In addition, we investigate the effects of shRNAs on the development of somatic cell nuclear transfer (SCNT) embryos. These results show that shRNA causes adverse effects in vitro and in vivo and shRNA-induced disruption of the endogenous miRNA pathway may lead to the early lethality of shRNA-transgenic pigs. We firstly report abnormalities of the miRNA pathway in shRNA-transgenic animals, which may explain the early lethality of shRNA-transgenic pigs and has important implications for shRNA-transgenic animal preparation.
authors.affiliation: Jilin Provincial Key Laboratory of Animal Embryo Engineering, College of Animal Sciences, Jilin University, Changchun 130062, China; Department of Life Sciences, Northeast Forestry University, Harbin 150040, China; Institute of Military Veterinary, Academy of Military Medical Sciences, Changchun 130062, China.
authors.forename: Zhen
authors.initials: Z
authors.lastname: Dai
authors.forename: Rong
authors.initials: R
authors.lastname: Wu
authors.forename: Yi-cheng
authors.initials: YC
authors.lastname: Zhao
authors.forename: Kan-kan
authors.initials: KK
authors.lastname: Wang
authors.forename: Yong-ye
authors.initials: YY
authors.lastname: Huang
authors.forename: Xin
authors.initials: X
authors.lastname: Yang
authors.forename: Zi-cong
authors.initials: ZC
authors.lastname: Xie
authors.forename: Chang-chun
authors.initials: CC
authors.lastname: Tu
authors.forename: Hong-sheng
authors.initials: HS
authors.lastname: Ouyang
authors.forename: Tie-dong
authors.initials: TD
authors.lastname: Wang
authors.forename: Da-xin
authors.initials: DX
authors.lastname: Pang
mesh_terms: D000818:Animals; D030801:Animals, Genetically Modified / Q000235:genetics*; D006692:Classical Swine Fever Virus / Q000235:genetics*; D012333:RNA, Messenger / Q000235:genetics*; D034741:RNA, Small Interfering / Q000235:genetics*; D015398:Signal Transduction / Q000235:genetics*; D015996:Survival Rate; D013552:Swine / Q000235:genetics*
chemical_list: D012333:RNA, Messenger; D034741:RNA, Small Interfering
keywords: Blastocyst formation; Classical swine fever virus (CSFV); Early lethality; MicroRNA pathway; shRNA-transgenic pigs