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.

Keywords: Blastocyst formation; Classical swine fever virus (CSFV); Early lethality; MicroRNA pathway; shRNA-transgenic pigs.

Fig. 1

Generation of shRNA-positive PFF clones (a, b) siRNA expression vectors PGKneolox2-shRNA (shN1, shN2, shNS3, shNS5, and shscrControl) and genomic structure and encoding proteins of the CSFV Shimen strain; (c) Overexpressed siRNA in clonal cells compared with scrambled shRNA-positive clonal cells (shscrControl), as determined by real-time PCR (data are expressed as mean±SD, n=6; P<0.05); (d) An image of a shRNA-positive PFF clone

Fig. 2

Protective effect of shRNA against CSFV infection Viral infection in PFF clones was examined by an IFA. Only a few cells in the antiviral shRNA wells displayed green fluorescence compared with the control, indicating that the shRNA inhibits CSFV effectively

Fig. 3

Adverse effects induced by shRNA in PFFs (a) IFN responsive genes (OASI, IFN-β) in clones increased compared with non-transfected control (P<0.05); (b) shRNA clones exhibited a little decreased proliferation compared to the non-transfected control cells; (c) miRNA processing enzyme Dicer and Drosha mRNA increased 50–250-fold in shRNA-positive cells (P<0.05); (d) Mature miRNAs including miR-21 and miR-196 in clonal cells increased significantly compared with non-transfected control (P<0.05). Data are expressed as mean±SD (n=6)

Fig. 4

Abnormalities of miRNA pathway in the livers of transgenic pigs (a) Overexpressed siRNA in transgenic animal liver tissue compared with normal liver tissue (tControl), as determined by real-time PCR (P<0.05); (b) All the miRNA processing enzymes expressed at a high level in the liver tissue of transgenic pigs (P<0.05); (c) More endogenous ubiquitous miRNAs, including miR-21, miR-30, miR-122, and miR-196, were detected and showed an increasing expression in livers of shRNA transgenic pigs (P<0.05); (d) Western blotting analysis of the AGO2 protein and Exportin5 protein in liver tissue. AGO2 decreased sharply in tissue samples in two replicates compared with the control (tControl), while Exportin5 decreased slightly. Data are expressed as mean±SD (n=6)