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Background: Pigs have emerged as one of the most popular large animal models in biomedical research, which in many cases is considered as a superior choice over rodent models. In addition, transplantation studies using pig pluripotent stem (PS) cell derivatives may serve as a testbed for safety and efficacy prior to human trials. Recently, it has been shown that mouse and human PS cells cultured in LCDM (recombinant human LIF, CHIR 99021, (S)-(+)-dimethindene maleate, minocycline hydrochloride) medium exhibited extended developmental potential (designated as extended pluripotent stem cells, or EPS cells), which could generate both embryonic and extraembryonic tissues in chimeric mouse conceptus. Whether stable pig induced pluripotent stem (iPS) cells can be generated in LCDM medium and their chimeric competency remains unknown. Methods: iPS cells were generated by infecting pig pericytes (PC) and embryonic fibroblasts (PEFs) with a retroviral vector encoding Oct4, Sox2, Klf4, and cMyc reprogramming factors and subsequently cultured in a modified LCDM medium. The pluripotency of PC-iPS and PEF-iPS cells was characterized by examining the expression of pluripotencyrelated transcription factors and surface markers, transcriptome analysis, and in vitro and in vivo differentiation capabilities. Chimeric contribution of PC-iPS cells to mouse and pig conceptus was also evaluated with fluorescence microscopy, flow cytometry, and PCR analysis.

BackgroundTransmissible gastroenteritis virus (TGEV) infection can activate NF-κB pathway in porcine intestinal epithelial cells and result in severe inflammation. Non-coding RNAs (ncRNAs) are not translated into proteins and play an important role in many biological and pathological processes such as inflammation, viral infection, and mitochondrial damage. However, whether ncRNAs participate in TGEV-induced inflammation in porcine intestinal epithelial cells is largely unknown.ResultsIn this study, the next-generation sequencing (NGS) technology was used to analyze the profiles of mRNAs, miRNAs, and circRNAs in Mock- and TGEV-infected intestinal porcine epithelial cell-jejunum 2 (IPEC-J2) cell line. A total of 523 mRNAs, 65 microRNAs (miRNAs), and 123 circular RNAs (circRNAs) were differentially expressed. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis showed differentially expressed mRNAs were linked to inflammation-related pathways, including NF-κB, Toll-like receptor, NOD-like receptor, Jak-STAT, TNF, and RIG-I-like receptor pathways. The interactions among mRNA, miRNA, and circRNA were analyzed. The data showed that ssc_circ_009380 and miR-22 might have interaction relationship. Dual-luciferase reporter assay confirmed that miR-22 directly bound to ssc_circ_009380. We also observed that overexpression of miR-22 led to a reduction of p-IκB-α and accumulation of p65 in nucleus in TGEV-infected IPEC-J2 cells. In contrast, inhibition of miR-22 had the opposite effects. Moreover, silencing of ssc_circ_009380 inhibited accumulation of p65 in nucleus and phosphorylation of IκB-α.ConclusionsThe data revealed that differentially expressed mRNAs and ncRNAs were primarily enriched in inflammation-related pathways and ssc_circ_009380 promoted activation of NF-κB pathway by binding miR-22 during TGEV-induced inflammation.Electronic supplementary materialThe online version of this article (10.1186/s12864-018-5128-5) contains supplementary material, which is available to authorized users.

Transgenic sheep can be used to achieve genetic improvements in breeds and as an important large-animal model for biomedical research. In this study, we generated a TALEN plasmid specific for ovine MSTN and transfected it into fetal fibroblast cells of STH sheep. MSTN biallelic-KO somatic cells were selected as nuclear donor cells for SCNT. In total, cloned embryos were transferred into 37 recipient gilts, 28 (75.7%) becoming pregnant and 15 delivering, resulting in 23 lambs, 12 of which were alive. Mutations in the lambs were verified via sequencing and T7EI assay, and the gene mutation site was consistent with that in the donor cells. Off-target analysis was performed, and no off-target mutations were detected. MSTN KO affected the mRNA expression of MSTN relative genes. The growth curve for the resulting sheep suggested that MSTN KO caused a remarkable increase in body weight compared with those of wild-type sheep. Histological analyses revealed that MSTN KO resulted in muscle fiber hypertrophy. These findings demonstrate the successful generation of MSTN biallelic-KO STH sheep via gene editing in somatic cells using TALEN technology and SCNT. These MSTN mutant sheep developed and grew normally, and exhibited increased body weight and muscle growth.

Pig epiblast-derived pluripotent stem cells are considered to have great potential and broad prospects for human therapeutic model development and livestock breeding. Despite ongoing attempts since the 1990s, no stably defined pig epiblast-derived stem cell line has been established. Here, guided by insights from a large-scale single-cell transcriptome analysis of pig embryos from embryonic day (E) 0 to E14, specifically, the tracing of pluripotency changes during epiblast development, we developed an in vitro culture medium for establishing and maintaining stable pluripotent stem cell lines from pig E10 pregastrulation epiblasts (pgEpiSCs). Enabled by chemical inhibition of WNT-related signaling in combination with growth factors in the FGF/ERK, JAK/STAT3, and Activin/Nodal pathways, pgEpiSCs maintain their pluripotency transcriptome features, similar to those of E10 epiblast cells, and normal karyotypes after more than 240 passages and have the potential to differentiate into three germ layers. Strikingly, ultradeep in situ Hi-C analysis revealed functional impacts of chromatin 3D-spatial associations on the transcriptional regulation of pluripotency marker genes in pgEpiSCs. In practice, we confirmed that pgEpiSCs readily tolerate at least three rounds of successive gene editing and generated cloned gene-edited live piglets. Our findings deliver on the long-anticipated promise of pig pluripotent stem cells and open new avenues for biological research, animal husbandry, and regenerative biomedicine.

Transmissible gastroenteritis virus (TGEV), a member of the coronaviridae family, could cause fatal diarrhea of piglets and result in numerous economic losses. Previous studies demonstrated that TGEV infection could lead to mitochondrial damage and upregulate miR-4331 level. So miR-4331 may play an important regulatory role in the control of mitochondrial function. To explore the potential role of miR-4331 in mitochondrial damage, we adopted a strategy consisting of quantitative proteomic analysis of porcine kidney (PK-15) cells in response to miR-4331 and TGEV infection. Eventually, 69 differentially expressed proteins were gained. The target of miR-4331 was identified. The effects of miR-4331 and its target RB1 on mitochondrial Ca2+ level, mitochondrial membrane potential (MMP), interleukin-1 receptor accessory protein (IL1RAP), p38 MAPK signaling pathway were investigated. The results showed that miR-4331 elevated mitochondrial Ca2+ level, reduced MMP, targets Retinoblastoma 1 (RB1), upregulated IL1RAP, and induced activation of p38 MAPK pathway during TGEV infection. RB1 was identified as the direct targets of miR-4331 and downregulated IL1RAP, suppressed the activation of p38 MPAK, and attenuated TGEV-induced mitochondrial damage. In addition, IL1RAP played a positive role in activating p38 MAPK signaling and negative role in TGEV-induced mitochondrial damage. The data indicate that miR-4331 aggravates TGEV-induced mitochondrial damage by repressing expression of RB1, promoting IL1RAP, and activating p38 MAPK pathway.

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Pig-to-human organ transplantation has drawn attention in recent years due to the potential use of pigs as an alternative source of human donor organs. While GGTA1 knockout (GTKO) can protect xenografts from hyperacute rejection, complement-dependent cytotoxicity might still contribute to this type of rejection. To prolong the xenograft survival, we utilized a T2A-mediated pCMV-hCD55-T2A-hCD59-Neo vector and transfected the plasmid into GTKO Diannan miniature pig fetal fibroblasts. After G418 selection combined with single-cell cloning culture, four colonies were obtained, and three of these were successfully transfected with the hCD55 and hCD59. One of the three colonies was selected as donor cells for somatic cell nuclear transfer (SCNT). Then, the reconstructed embryos were transferred into eight recipient gilts, resulting in four pregnancies. Three of the pregnant gilts delivered, yielding six piglets. Only one piglet carried hCD55 and hCD59 genetic modification. The expression levels of the GGTA1, hCD55, and hCD59 in the tissues and fibroblasts of the piglet were determined by q-PCR, fluorescence microscopy, immunohistochemical staining, and western blotting analyses. The results showed the absence of GGTA1 and the coexpression of the hCD55 and hCD59. However, the mRNA expression levels of hCD55 and hCD59 in the GTKO/hCD55/hCD59 pig fibroblasts were lower than that in human 293T cells, which may be caused by low copy number and/or CMV promoter methylation. Furthermore, we performed human complement-mediated cytolysis assays using human serum solutions from 0 to 60%. The result showed that the fibroblasts of this triple-gene modified piglet had greater survival rates than that of wild-type and GTKO controls. Taken together, these results indicate that T2A-mediated polycistronic vector system combined with SCNT can effectively generate multiplex genetically modified pigs, additional hCD55 and hCD59 expression on top of a GTKO genetic background markedly enhance the protective effect towards human serum-mediated cytolysis than those of GTKO alone. Thus, we suggest that GTKO/hCD55/hCD59 triple-gene-modified Diannan miniature pig will be a more eligible donor for xenotransplantation.