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Fan A.,Center for Animal Embryo Engineering of Jilin Province | Ma K.,Center for Animal Embryo Engineering of Jilin Province | An X.,Center for Animal Embryo Engineering of Jilin Province | Ding Y.,Jilin University | And 9 more authors.
Reproduction | Year: 2013

TET1 is implicated in maintaining the pluripotency of embryonic stem cells. However, its precise effects on induced pluripotent stem cells (iPSCs), and particularly on porcine iPSCs (piPSCs), are not well defined. To investigate the role of TET1 in the pluripotency and differentiation of piPSCs, piPSCs were induced from porcine embryonic fibroblasts by overexpression of POU5F1(OCT4), SOX2, KLF4, and MYC (C-MYC). siRNAs targeting to TET1 were used to transiently knockdown the expression of TET1 in piPSCs. Morphological abnormalities and loss of the undifferentiated state of piPSCs were observed in the piPSCs after the downregulation of TET1. The effects of TET1 knockdown on the expression of key stem cell factors and differentiation markers were analyzed to gain insights into the molecular mechanisms underlying the phenomenon. The results revealed that knockdown of TET1 resulted in the downregulated expression of pluripotency-related genes, such as LEFTY2, KLF2, and SOX2, and the upregulated expression of differentiation-related genes including PITX2, HAND1, GATA6, and LEF1. However, POU5F1, MYC, KLF4, and NANOG were actually not downregulated. Further analysis showed that the methylation levels of the promoters for POU5F1 and MYC increased significantly after TET1 downregulation, whereas there were no obvious changes in the promoters of SOX2, KLF4, and NANOG. The methylation of the whole genome increased, while hydroxymethylation slightly declined. Taken together, these results suggest that TET1 may play important roles in the self-renewal of piPSCs and the maintenance of their characteristics by regulating the expression of genes and the DNA methylation. © 2013 Society for Reproduction and Fertility. Source


Lan Y.,Jilin University | Lu H.,Jilin University | Zhao K.,Jilin University | He W.,Jilin University | And 6 more authors.
Intervirology | Year: 2012

Objective: The specific effect of RNA interference on the replication of porcine hemagglutinating encephalomyelitis virus (PHE-CoV) was explored. Methods: Four species of small interfering RNA (siRNA), targeting different regions of the PHE-CoV spike glycoprotein and replicase polyprotein genes, were prepared by in vitro transcription. After transfection of PK-15 cells with each of the siRNAs followed by infection with PHE-CoV, the cytopathic effect (CPE) was examined by phase-contrast microscope, and viral proliferation within cells was examined by indirect immunofluorescence microscopy, hemagglutination (HA) test, TCID 50 assay and real-time RT-PCR. Results: Examination of CPE demonstrated that the four siRNAs were capable of protecting cells against PHE-CoV invasion with very high specificity and efficiency. At 48 h post-infection, only a few siRNA-treated cells were positive for viral antigen staining, whereas most untreated virus-infected cells were positive. Transfection with siRNAs also suppressed the production of infectious virus by up to 18-to 32-fold as assessed by a HA test and 93-to 494-fold as assessed by TCID 50 assay. Furthermore, treatment with siRNAs caused a 53-91% reduction in the viral genome copy number as assessed by real-time RT-PCR. Conclusion: These results suggested that the four species of siRNAs can efficiently inhibit PHE-CoV genome replication and infectious virus production. Copyright © 2011 S. Karger AG, Basel. Source

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