Transgenic pigs play an important role in producing higher quality food in agriculture and improving human health when used as animal models for various human diseases in biomedicine. Production of transgenic pigs, however, is a lengthy and inefficient process that hinders research using pig models. Recent applications of the CRISPR/Cas9 system for generating site-specific gene knockout/knockin models, including a knockout pig model, have significantly accelerated the animal model field. However, a knockin pig model containing a site-specific transgene insertion that can be passed on to its offspring remains lacking. Here, we describe for the first time the generation of a site-specific knockin pig model using a combination of CRISPR/Cas9 and somatic cell nuclear transfer. We also report a new genomic “safe harbor” locus, named pH11, which enables stable and robust transgene expression. Our results indicate that our CRISPR/Cas9 knockin system allows highly efficient gene insertion at the pH11 locus of up to 54% using drug selection and 6% without drug selection. We successfully inserted a gene fragment larger than 9 kb at the pH11 locus using the CRISPR/Cas9 system. Our data also confirm that the gene inserted into the pH11 locus is highly expressed in cells, embryos and animals.

Long-term adherence to a high-fat, high-calorie diet influences human health and causes obesity, metabolic syndrome and nonalcoholic steatohepatitis (NASH). Inflammation plays a key role in the development of NASH; however, the mechanism of inflammation induced by over-nutrition remains largely unknown. In this study, we fed Bama minipigs a high-fat, high-sucrose diet (HFHSD) for 23 months. The pigs exhibited characteristics of metabolic syndrome and developed steatohepatitis with greatly increased numbers of inflammatory cells, such as lymphocytes (2.27-fold, P<0.05), Kupffer cells (2.59-fold, P<0.05), eosinophils (1.42-fold, P<0.05) and neutrophils (2.77-fold, P<0.05). High-throughput RNA sequencing (RNA-seq) was performed to explore the systemic transcriptome of the pig liver during inflammation. Approximately 18.2 gigabases of raw sequence data were generated, and over 303 million high-quality reads were assembled into 21,126 unigenes. RNA-seq data analysis showed that 822 genes were differentially expressed in liver (P<0.05) between the HFHSD and control groups. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis showed that the process of inflammation involved the inflammatory signal transduction-related toll-like receptor, MAPK, and PPAR signaling pathways; the cytokine-related chemokine signaling, cytokine-cytokine receptor interaction, and IL2, IL4, IL6, and IL12 signaling pathways; the leukocyte receptor signaling-related T cell, B cell, and natural killer cell signaling pathways; inflammatory cell migration and invasion- related pathways; and other pathways. Moreover, we identified several differentially expressed inflammation-related genes between the two groups, including FOS, JUN, TLR7, MYC, PIK3CD, VAV3, IL2RB and IL4R, that could be potential targets for further investigation. Our study suggested that long-term HFHSD induced obesity and liver inflammation, providing basic insight into the molecular mechanism of this condition and laying the groundwork for further studies on obesity and steatohepatitis.

Metabolic syndrome can induce chronic renal injury in humans. In the present study, Bama minipigs were fed a high-fat/high-sucrose diet (HFHSD) for 23 months, which caused them to develop the pathological characteristics of metabolic syndrome, including obesity, hyperinsulinemia, and hyperlipidemia, and resulted in kidney tissue damage. In the HFHSD group, the ratio of the glomus areas to the glomerulus area and the glomerular density inside the renal cortex both decreased. Lipid deposition in the renal tubules was detected in the HFHSD group, and up-regulated expression levels of SREBP-1, FABP3 and LEPR promoted lipid deposition. The decreased levels of SOD, T-AOC and GSH-PX indicated that the antioxidant capacity of the renal tissues was diminished in the HFHSD group compared with MDA, which increased. The renal tissue in the HFHSD group exhibited clear signs of inflammation as well as significantly elevated expression of key genes associated with inflammation, including tumor necrosis factor-α (TNF-α) and macrophage migration inhibitory factor (MIF), compared with the control group. The tubular epithelial cells in the HFHSD group displayed significantly greater numbers of apoptotic cells, and the expression of proliferating cell nuclear antigen (PCNA) in the renal tubules decreased. Caspase-3 expression increased significantly, and the transcription factor nuclear factor κB (NF-κB) was activated and translocated into the nucleus. In conclusion, long-term HFHSDs cause metabolic syndrome and chronic renal tissue injury in Bama minipigs. These findings provide a foundation for further studies investigating metabolic syndrome and nephropathy.

A long-term high-energy diet affects human health and leads to obesity and metabolic syndrome in addition to cardiac steatosis and hypertrophy. Ectopic fat accumulation in the heart has been demonstrated to be a risk factor for heart disorders, but the molecular mechanism of heart disease remains largely unknown. Bama miniature pigs were fed a high-fat, high-sucrose diet (HFHSD) for 23 months. These pigs developed symptoms of metabolic syndrome and showed cardiac steatosis and hypertrophy with a greatly increased body weight (2.73-fold, P<0.01), insulin level (4.60-fold, P<0.01), heart weight (1.82-fold, P<0.05) and heart volume (1.60-fold, P<0.05) compared with the control pigs. To understand the molecular mechanisms of cardiac steatosis and hypertrophy, nine pig heart cRNA samples were hybridized to porcine GeneChips. Microarray analyses revealed that 1,022 genes were significantly differentially expressed (P<0.05, ≥1.5-fold change), including 591 up-regulated and 431 down-regulated genes in the HFHSD group relative to the control group. KEGG analysis indicated that the observed heart disorder involved the signal transduction-related MAPK, cytokine, and PPAR signaling pathways, energy metabolism-related fatty acid and oxidative phosphorylation signaling pathways, heart function signaling-related focal adhesion, axon guidance, hypertrophic cardiomyopathy and actin cytoskeleton signaling pathways, inflammation and apoptosis pathways, and others. Quantitative RT-PCR assays identified several important differentially expressed heart-related genes, including STAT3, ACSL4, ATF4, FADD, PPP3CA, CD74, SLA-8, VCL, ACTN2 and FGFR1, which may be targets of further research. This study shows that a long-term, high-energy diet induces obesity, cardiac steatosis, and hypertrophy and provides insights into the molecular mechanisms of hypertrophy and fatty heart to facilitate further research.

Introduction: Mesenchymal stem cells (MSCs) have the ability to proliferate in vivo with a large variety of differentiation potentials and therefore are widely used as an ideal material for cell therapy. MSCs derived from pig and human sources are similar in many aspects, such as cell immunophenotype and functional characteristics. However, differences in proteomics and the molecular mechanisms of cell functions between porcine bone marrow MSCs (BM-MSCs) and umbilical cord MSCs (UC-MSCs) are largely unknown. To the best of our knowledge, MSCs collected from different tissue have specific phenotype and differentiation ability in response to microenvironment, known as a niche. Methods: Porcine BM-MSCs and UC-MSCs were evaluated with flow cytometric and adipogenic and osteogenic differentiation analyses. We used isobaric tagging for relative and absolute quantitation (iTRAQ), combined with liquid chromatography-tandem mass spectrometry, to identify differentially expressed proteins (DEPs) between these two types of MSCs. Kyoto Encyclopedia of Genes and Genomes pathway and phenotype analyses were used to understand the links between cell migration ability and DEPs. Results: Two separate iTRAQ experiments were conducted, identifying 95 DEPs (95% confidence interval). Five of these proteins were verified by Western blotting. These 95 DEPs were classified in terms of biological regulation, metabolic process, developmental process, immune system process, reproduction, death, growth, signaling, localization, response to stimulus, biological adhesion, and cellular component organization. Our study is the first to show results indicating that porcine BM-MSCs have a higher migration capability than UC-MSCs. Finally, one of the DEPs, Vimentin, was verified to have a positive role in MSC migration. Conclusions: These results represent the first attempt to use proteomics specifically targeted to porcine MSCs of different tissues. The identified components should help reveal a variety of tissue-specific functions in tissue-derived MSC populations and could serve as important tools for the regeneration of particular tissues in future stem cell-based tissue engineering studies using animal models.

Today, obesity and nonalcoholic steatohepatitis are a worldwide epidemic, although how these syndromes are regulated with respect to lncRNAs remains largely unknown. Our previous studies have revealed important pathological features and molecular characteristics of nonalcoholic steatohepatitis in the minipig model, and in this study, we analyze the features of lncRNAs and their potential target genes. Minipig samples only from liver were analyzed using next-generation deep sequencing. In total, we obtained 585 million raw reads approximately 70.4 Gb of high quality data. After a strict five-step filtering process, 1,179 lncRNAs were identified, including 89 differentially expressed lncRNAs (P < 0.05) in the experiment group relative to the control group. The cis and trans analysis identified target genes that were enriched for specific GO terms (P < 0.01), including immune processes, chemokine activity, cytokine activity, and G-protein coupled receptor binding, which are closely related to nonalcoholic steatohepatitis. The predicted protein-coding targets of the differentially expressed lncRNAs were further analyzed, such as PPAR, FADS2, DGAT2, ACAA2, CYP2E1, ADH4, and Fos. This study reveals a wealth of candidate lncRNAs involved in NASH and their regulated pathways, which should facilitate further research into the molecular mechanisms of this disorder.