Comparative analysis of swine leukocyte antigen gene diversity in Göttingen Minipigs.

Worldwide, pigs represent economically important farm animals, also representing a preferred preclinical large animal model for biomedical studies. The need for swine leukocyte antigen (SLA) typing is increasing with the expanded use of pigs in translational research, infection studies, and for veterinary vaccine design. Göttingen Minipigs (GMP) attract increasing attention as valuable model for pharmacological studies and transplantation research. This study represents a first-time assessment of the SLA gene diversity in Göttingen Minipigs in combination with a comparative metadata analysis with commercial pig lines. As Göttingen Minipigs could harbor private as well as potential novel SLA allele combinations, future research projects would benefit from the characterization of their SLA background. In 209 Göttingen Minipigs, SLA class I (SLA-1, SLA-2, SLA-3) and class II (DRB1, DQB1, DQA) genes were characterized by PCR-based low-resolution (Lr) haplotyping. Criteria and nomenclature used for SLA haplotyping were proposed by the ISAG/IUIS-VIC SLA Nomenclature Committee. Haplotypes were assigned based on the comparison with already known breed or farm-specific allele group combinations. In total, 14 SLA class I and five SLA class II haplotypes were identified in the studied cohort, to manifest in 26 SLA class I but only seven SLA class II genotypes. The most common SLA class I haplotypes Lr-24.0 (SLA-1*15XX or Blank-SLA-3*04:04-SLA-2*06:01~02) and Lr-GMP-3.0 (SLA-1*16:02-SLA-3*03:04-SLA-2*17:01) occurred at frequencies of 23.44 and 18.66%, respectively. For SLA class II, the most prevalent haplotypes Lr-0.21 (DRB1*01XX-DQB1*05XX-DQA*04XX) and Lr-0.03 (DRB1*03:02-DQB1*03:01-DQA*01XX) occurred at frequencies of 38.28 and 30.38%. The comparative metadata analysis revealed that Göttingen Minipigs only share six SLA class I and two SLA class II haplotypes with commercial pig lines. More importantly, despite the limited number of SLA class I haplotypes, the high genotype diversity being observed necessitates pre-experimental SLA background assessment of Göttingen Minipigs in regenerative medicine, allo-transplantation, and xenograft research.

Integrated analysis of lncRNA, miRNA and mRNA expression profiles reveals regulatory pathways associated with pig testis function.

Long noncoding RNA (lncRNA) and microRNA (miRNA) are known to play pivotal roles in mammalian testicular function and spermatogenesis. However, their impact on porcine male reproduction has yet to be well unraveled. Here, we sequenced and identified lncRNA and miRNA expressed in the testes of Chinese indigenous Banna mini-pig inbred line (BMI) and introduced Western Duroc (DU) and Large White (LW) pigs. By pairwise comparison (BMI vs DU, BMI vs LW, and DU vs LW), we found the gene expression differences in the testes between Chinese local pigs and introduced Western commercial breeds were more striking than those between introduced commercial breeds. Furthermore, we found 1622 co-differentially expressed genes (co-DEGs), 122 co-differentially expressed lncRNAs (co-DELs), 39 co-differentially expressed miRNAs (co-DEMs) in BMI vs introduced commercial breeds (DU and LW). Functional analysis revealed that these co-DEGs and co-DELs/co-DEMs target genes were enriched in male sexual function pathways, including MAPK, AMPK, TGF-ß/Smad, Hippo, NF-kappa B, and PI3K/Akt signaling pathways. Additionally, we established 10,536 lncRNA-mRNA, 11,248 miRNA-mRNA pairs, and 62 ceRNA (lncRNA-miRNA-mRNA) networks. The ssc-miR-1343 had the most interactive factors in the ceRNA network, including 20 mRNAs and 3 lncRNAs, consisting of 56 ceRNA pairs. These factors played extremely important roles in the regulation of testis function as key nodes in the interactive regulatory network. Our results provide insight into the functional roles of lncRNAs and miRNAs in porcine testis and offer a valuable resource for understanding the differences between Chinese indigenous and introduced Western pigs.

Analysis of production efficiency of cloned transgenic Yucatan miniature pigs according to recipient breeds with embryo transfer conditions.

The purpose of this study was to compare the efficiency of the production of cloned transgenic Yucatan miniature pigs (YMPs) using two recipient breeds, i.e., YMPs and domestic pigs (DPs), under various embryo transfer conditions. We initially assessed the in vitro developmental competence of embryos obtained via somatic cell nuclear transfer (SCNT) from three different transgenic donor cells. No difference was observed among the three groups regarding developmental competence. Furthermore, the cloning efficiency remained consistent among the three groups after the transfer of the SCNT embryos to each surrogate mother. Subsequently, to compare the efficiency of the production of cloned transgenic YMPs between the two recipient breeds using varying parameters, including ovulation status (preovulation and postovulation), duration of in vitro culture (IVC) (incubated within 24 h and 24-48 h), and the number of transferred SCNT embryos (less than and more than 300), we assessed the pregnancy rates, delivery rates, mean offspring counts, and cloning efficiency. Regarding the ovulation status, YMPs exhibited higher pregnancy rates, delivery rates, and cloning efficiency compared with DPs in both statuses. Moreover, the pregnancy rates, delivery rates, and cloning efficiency were affected by the ovulation status in DPs, but not in YMPs. The comparison of IVC duration between groups revealed that YMPs had higher pregnancy rates vs. DPs in both conditions. SCNT embryos cultured for 24-48 h in YMPs yielded higher delivery rates and cloning efficiency compared with those cultured for less than 24 h in DPs. Finally, the analysis based on the number of transferred SCNT embryos showed that both the pregnancy and delivery rates were higher in YMPs vs. DPs. However, the highest average number of offspring was obtained when more than 300 SCNT embryos were transferred into DPs, whereas the cloning efficiency was higher in YMPs vs. DPs. Our results suggest that YMPs are more suitable recipients than are DPs under various conditions for the production of cloned transgenic YMPs.

Differences in vasomotor function of mesenteric arteries between Ossabaw minipigs with predisposition to metabolic syndrome and Göttingen minipigs.

Metabolic syndrome predisposes and contributes to the development and progression of atherosclerosis. The minipig strain "Ossabaw" is characterized by a predisposition to develop metabolic syndrome. We compared vasomotor function in Ossabaw minipigs before they developed their diseased phenotype to that of Göttingen minipigs without such genetic predisposition. Mesenteric arteries of adult Ossabaw and Göttingen minipigs were dissected postmortem and mounted on a myograph for isometric force measurements. Maximal vasoconstriction to potassium chloride (KClmax) was induced. Cumulative concentration-response curves were determined in response to norepinephrine. Endothelium-dependent (with carbachol) and endothelium-independent (with nitroprusside) vasodilation were analyzed after preconstriction by norepinephrine. In a bioinformatic analysis, variants/altered base pairs within genes associated with cardiovascular disease were analyzed. KClmax was similar between the minipig strains (15.6 ± 6.7 vs. 14.1 ± 3.4 ΔmN). Vasoconstriction in response to norepinephrine was more pronounced in Ossabaw than in Göttingen minipigs (increase of force to 143 ± 48 vs. 108 ± 38% of KClmax). Endothelium-dependent and endothelium-independent vasodilation were less pronounced in Ossabaw than in Göttingen minipigs (decrease of force to 46.4 ± 29.6 vs. 16.0 ± 18.4% and to 36.7 ± 25.2 vs. 2.3 ± 3.7% of norepinephrine-induced preconstriction). Vasomotor function was not different between the sexes. More altered base pairs/variants were identified in Ossabaw than in Göttingen minipigs for the exon encoding adrenoceptor-α1A. Vasomotor function in lean Ossabaw minipigs is shifted toward vasoconstriction and away from vasodilation in comparison with Göttingen minipigs, suggesting a genetic predisposition for vascular dysfunction and atherosclerosis in Ossabaw minipigs. Thus, Ossabaw minipigs may be a better model for human cardiovascular disease than Göttingen minipigs.NEW & NOTEWORTHY Animal models with a predisposition to metabolic syndrome and atherosclerosis are attracting growing interest for translational research, as they may better mimic the variability of patients with cardiovascular disease. In Ossabaw minipigs, with a polygenic predisposition to metabolic syndrome, but without the diseased phenotype, vasoconstriction is more and vasodilation is less pronounced in mesenteric arteries than in Göttingen minipigs. Ossabaw minipigs may be a more suitable model of human cardiovascular disease.

Longitudinal dynamics of circulating miRNAs in a swine model of familial hypercholesterolemia during early atherosclerosis.

Atherosclerosis is a complex progressive disease involving intertwined biological mechanisms. We aimed to identify miRNA expression dynamics at the early stages of atherosclerosis using a large swine model (Wisconsin Miniature Swine, WMS). A total of 18 female pigs; 9 familial hypercholesterolemic (WMS-FH) and 9 normal control swine (WMS-N) were studied. miRNA sequencing was performed on plasma cell-free RNA at 3, 6, and 9 months of age. RT-qPCR validated DE miRNAs in a new cohort of animals (n = 30) with both sexes. Gene ontology and mRNA targets for DE miRNAs were identified. In vivo multimodality imaging and histopathology were performed to document the presence of atherosclerosis at termination. 20, 19, and 9 miRNAs were significantly DE between the groups at months 3, 6, and 9, respectively. Most DE miRNAs and their target genes are involved in human atherosclerosis development. Coronary atherosclerosis was documented in 7/9 WMS-FH pigs. Control animals had no lesions. miR-138, miR-152, miR-190a, and miR-196a showed a significant diagnostic power at month 3, whereas miR-486, miR-126-3p, miR-335, and miR-423-5p were of significant diagnostic power at month 9. In conclusion, specific DE miRNAs with significant discriminatory power may be promising biomarkers for the early detection of coronary atherosclerosis.

Early to sustained impacts of lethal radiation on circulating miRNAs in a minipig model.

Early diagnosis of lethal radiation is imperative since its intervention time windows are considerably short. Hence, ideal diagnostic candidates of radiation should be easily accessible, enable to inform about the stress history and objectively triage subjects in a time-efficient manner. Therefore, the small molecules such as metabolites and microRNAs (miRNAs) from plasma are legitimate biomarker candidate for lethal radiation. Our objectives were to comprehend the radiation-driven molecular pathogenesis and thereby determine biomarkers of translational potential. We investigated an established minipig model of LD70/45 total body irradiation (TBI). In this pilot study, plasma was collected pre-TBI and at multiple time points post-TBI. The majority of differentially expressed miRNAs and metabolites were perturbed immediately after TBI that potentially underlined the severity of its acute impact. The integrative network analysis of miRNA and metabolites showed a cohesive response; the early and consistent perturbations of networks were linked to cancer and the shift in musculoskeletal atrophy synchronized with the comorbidity-networks associated with inflammation and bioenergy synthesis. Subsequent comparative pipeline delivered 92 miRNAs, which demonstrated sequential homology between human and minipig, and potentially similar responses to lethal radiation across these two species. This panel promised to retrospectively inform the time since the radiation occurred; thereby could facilitate knowledge-driven interventions.

Nano-Architecture of Persistent Focal DNA Damage Regions in the Minipig Epidermis Weeks after Acute γ-Irradiation.

Exposure to high acute doses of ionizing radiation (IR) can induce cutaneous radiation syndrome. Weeks after such radiation insults, keratinocyte nuclei of the epidermis exhibit persisting genomic lesions that present as focal accumulations of DNA double-strand break (DSB) damage marker proteins. Knowledge about the nanostructure of these genomic lesions is scarce. Here, we compared the chromatin nano-architecture with respect to DNA damage response (DDR) factors in persistent genomic DNA damage regions and healthy chromatin in epidermis sections of two minipigs 28 days after lumbar irradiation with ~50 Gy γ-rays, using single-molecule localization microscopy (SMLM) combined with geometric and topological mathematical analyses. SMLM analysis of fluorochrome-stained paraffin sections revealed, within keratinocyte nuclei with perisitent DNA damage, the nano-arrangements of pATM, 53BP1 and Mre11 DDR proteins in γ-H2AX-positive focal chromatin areas (termed macro-foci). It was found that persistent macro-foci contained on average ~70% of 53BP1, ~23% of MRE11 and ~25% of pATM single molecule signals of a nucleus. MRE11 and pATM fluorescent tags were organized in focal nanoclusters peaking at about 40 nm diameter, while 53BP1 tags formed nanoclusters that made up super-foci of about 300 nm in size. Relative to undamaged nuclear chromatin, the enrichment of DDR protein signal tags in γ-H2AX macro-foci was on average 8.7-fold (±3) for 53BP1, 3.4-fold (±1.3) for MRE11 and 3.6-fold (±1.8) for pATM. The persistent macro-foci of minipig epidermis displayed a ~2-fold enrichment of DDR proteins, relative to DSB foci of lymphoblastoid control cells 30 min after 0.5 Gy X-ray exposure. A lasting accumulation of damage signaling and sensing molecules such as pATM and 53BP1, as well as the DSB end-processing protein MRE11 in the persistent macro-foci suggests the presence of diverse DNA damages which pose an insurmountable problem for DSB repair.

Integration of 16S rRNA gene sequencing and LC/MS-based metabolomic analysis of early biomarkers of acute ischaemic stroke in Tibetan miniature pigs.

Acute ischaemic stroke (AIS) is a complex, systemic, pathological, and physiological process. Systemic inflammatory responses and disorders of the gut microbiome contribute to increased mortality and disability following AIS. We conducted 16S high-throughput sequencing and ultra-performance liquid chromatography-quadrupole time-of-flight tandem mass spectrometry-based non-targeted metabolomic analyses of the plasma from a Tibetan miniature pig middle cerebral artery occlusion (MCAO) model. A significant decrease in the abundance of Firmicutes and a significant increase in the abundance of Actinobacteria were observed after the onset of AIS. Among the plasma metabolites, the levels of phospholipids and amino acids were considerably altered. Loading values and differential metabolite-bacterial group association analyses of the metabolome and microbiome indicated a correlation between the microbiome and metabolome of Tibetan miniature pigs after MCAO. Furthermore, significant changes were observed in the ABC transporter pathway and purine metabolism in the gut microbiome-plasma metabolome during the early stage of AIS. Kyoto Encyclopaedia of Genes and Genomes enrichment analysis showed that arginine, proline, and cyanoamino acid metabolism was upregulated while ABC transporter metabolism pathway and carbohydrate digestion and absorption were substantially downregulated. The results of this study suggest that AIS affects the gut microbiota and plasma metabolites in Tibetan miniature pigs and that faecal microbiota transplantation could be a potential therapeutic approach for AIS.

Microscopic and transcriptomic changes in porcine synovium one year following disruption of the anterior cruciate ligament.

OBJECTIVE: There is no disease-modifying treatment for posttraumatic osteoarthritis (PTOA). This may be partly due to an incomplete understanding of synovitis, which has been causally linked to PTOA progression. The microscopic and transcriptomic changes in synovium seen in early- to mid-stage PTOA were evaluated to better characterize this knowledge gap. METHODS: Seventy-two Yucatan minipigs underwent transection of the anterior cruciate ligament (ACL). Subjects were randomized to no further intervention, ligament reconstruction, or ligament repair, followed by microscopic synovium evaluation and RNA-sequencing at 1, 4, and 52 weeks. Six additional subjects received no ligament transection and served as 1- and 4-week controls and 12 contralateral knees served as 52-week controls. RESULTS: Synovial lining thickness, stromal cellularity, and overall microscopic synovitis reached their highest levels in the first few weeks following injury. Inflammatory infiltration continued to increase over the course of a year. Leaving the ACL transected, reconstructing the ligament, or repairing the ligament did not modulate synovitis development at 1, 4, or 52 weeks. Differential gene expression analysis of PTOA-affected synovium compared to control synovium revealed increased cell proliferation, angiogenesis, collagen breakdown, and diminished lipid metabolism at 1 and 4 weeks, and increased axonogenesis and focal adhesion with reduced immune activation at 52 weeks. CONCLUSIONS: Synovitis was present one year after ACL injury and was not alleviated by surgical intervention. Gene expression in early synovitis was characterized by cell proliferation, angiogenesis, proteolysis, and reduced lipolysis, which was followed by nerve growth and cellular adhesion with less immune activation at 52 weeks.

Whole-genome sequencing study to identify candidate markers indicating susceptibility to type 2 diabetes in Bama miniature pigs.

BACKGROUND: Hundreds of single-nucleotide polymorphism (SNP) sites have been found to be potential genetic markers of type 2 diabetes mellitus (T2DM). However, SNPs related to T2DM in minipigs have been less reported. This study aimed to screen the T2DM-susceptible candidate SNP loci in Bama minipigs so as to improve the success rate of the minipig T2DM model. METHODS: The genomic DNAs of three Bama minipigs with T2DM, six sibling low-susceptibility minipigs with T2DM, and three normal control minipigs were compared by whole-genome sequencing. The T2DM Bama minipig-specific loci were obtained, and their functions were annotated. Meanwhile, the Biomart software was used to perform homology alignment with T2DM-related loci obtained from the human genome-wide association study to screen candidate SNP markers for T2DM in Bama miniature pigs. RESULTS: Whole-genome resequencing detected 6960 specific loci in the minipigs with T2DM, and 13 loci corresponding to 9 diabetes-related genes were selected. Further, a set of 122 specific loci in 69 orthologous genes of human T2DM candidate genes were obtained in the pigs. Collectively, a batch of T2DM-susceptible candidate SNP markers in Bama minipigs, covering 16 genes and 135 loci, was established. CONCLUSIONS: Whole-genome sequencing and comparative genomics analysis of the orthologous genes in pigs that corresponded to the human T2DM-related variant loci successfully screened out T2DM-susceptible candidate markers in Bama miniature pigs. Using these loci to predict the susceptibility of the pigs before constructing an animal model of T2DM may help to establish an ideal animal model.

Transcriptomic changes in porcine articular cartilage one year following disruption of the anterior cruciate ligament.

To determine the transcriptomic changes seen in early- to mid-stage posttraumatic osteoarthritis (PTOA) development, 72 Yucatan minipigs underwent transection of the anterior cruciate ligament. Subjects were randomized to no further intervention, ligament reconstruction, or ligament repair, followed by articular cartilage harvesting and RNA-sequencing at three different postoperative timepoints (1, 4, and 52 weeks). Six additional subjects received no ligament transection and provided cartilage tissue to serve as controls. Differential gene expression analysis between post-transection cartilage and healthy cartilage revealed an initial increase in transcriptomic differences at 1 and 4 weeks followed by a stark reduction in transcriptomic differences at 52 weeks. This analysis also showed how different treatments genetically modulate the course of PTOA following ligament disruption. Specific genes (e.g., MMP1, POSTN, IGF1, PTGFR, HK1) were identified as being upregulated in the cartilage of injured subjects across all timepoints regardless of treatment. At the 52-week timepoint, 4 genes (e.g., A4GALT, EFS, NPTXR, ABCA3) that-as far as we know-have yet to be associated with PTOA were identified as being concordantly differentially expressed across all treatment groups when compared to controls. Functional pathway analysis of injured subject cartilage compared to control cartilage revealed overarching patterns of cellular proliferation at 1 week, angiogenesis, ECM interaction, focal adhesion, and cellular migration at 4 weeks, and calcium signaling, immune system activation, GABA signaling, and HIF-1 signaling at 52 weeks.

Nanopore sequencing reveals methylation changes associated with obesity in circulating cell-free DNA from Göttingen Minipigs.

Profiling of circulating cell-free DNA (cfDNA) by tissue-specific base modifications, such as 5-methylcytosines (5mC), may enable the monitoring of ongoing pathophysiological processes. Nanopore sequencing allows genome-wide 5mC detection in cfDNA without bisulphite conversion. The aims of this study were: i) to find differentially methylated regions (DMRs) of cfDNA associated with obesity in Göttingen minipigs using Nanopore sequencing, ii) to validate a subset of the DMRs using methylation-specific PCR (MSP-PCR), and iii) to compare the cfDNA DMRs with those from whole blood genomic DNA (gDNA). Serum cfDNA and gDNA were obtained from 10 lean and 7 obese Göttingen Minipigs both with experimentally induced myocardial infarction and sequenced using Oxford Nanopore MinION. A total of 1,236 cfDNA DMRs (FDR<0.01) were associated with obesity. In silico analysis showed enrichment of the adipocytokine signalling, glucagon signalling, and cellular glucose homoeostasis pathways. A strong cfDNA DMR was discovered in PPARGC1B, a gene linked to obesity and type 2 diabetes. The DMR was validated using MSP-PCR and correlated significantly with body weight (P < 0.05). No DMRs intersected between cfDNA and gDNA, suggesting that cfDNA originates from body-wide shedding of DNA. In conclusion, nanopore sequencing detected differential methylation in minute quantities (0.1-1 ng/µl) of cfDNA. Future work should focus on translation into human and comparing 5mC from somatic tissues to pinpoint the exact location of pathology.

Oxford nanopore sequencing can reveal changes in methylation patterns associated with obesity in minute quantities of cell-free DNA from serum.Bisulphite conversion and methylation-specific PCR can be used to validate differentially methylated regions in cell-free DNA.A differentially methylated region in an intronic region of the PPARGC1B gene was found associated with obesity.Differentially methylated regions in cell-free DNA could be useful as early risk markers of certain diseases and pathologies.

Cloning of STC-1 and analysis of its differential expression in Hezuo pig.

The purpose of this study was to examine STC-1's structure, function, and differential expression in large and miniature pigs. We cloned the Hezuo pig's coding sequence, compared its homology, and used bioinformatics to assess the structure. RT-qPCR and Western blot were used to detect the expression in ten tissues of Hezuo pig and Landrace pig. The results showed that Hezuo pig was most closely related to Capra hircus and most distantly related to Danio rerio. The protein STC-1 has a signal peptide and its secondary structure is dominated by the alpha helix. The mRNA expression in the spleen, duodenum, jejunum, and stomach of Hezuo pigs was higher than that of Landrace pigs. And except for heart and duodenum, expression of the protein in Hezuo pig was higher than in another. In conclusion, STC-1 is highly conserved among different breeds of pigs, and the expression and distribution of its mRNA and protein are different in large and miniature pigs. This work can lay the foundation for future study into the mechanism of action of STC-1 in Hezuo pigs and the enhancement of breeding in miniature pigs.

Isolation of an Ecotropic Porcine Endogenous Retrovirus PERV-C from a Yucatan SLAD/D Inbred Miniature Swine.

Xenotransplantation may compensate the limited number of human allografts for transplantation using pigs as organ donors. Porcine endogenous retroviruses inherit infectious potential if pig cells, tissues, or organs were transplanted to immunosuppressed human recipients. Particularly, ecotropic PERV-C that could recombine with PERV-A to highly replication-competent human-tropic PERV-A/C should be excluded from pig breeds designed for xenotransplantation. Because of their low proviral background, SLAD/D (SLA, swine leukocyte antigen) haplotype pigs are potential candidates as organ donors as they do not bear replication-competent PERV-A and -B, even if they carry PERV-C. In this work, we characterized their PERV-C background isolating a full-length PERV-C proviral clone number 561 from a SLAD/D haplotype pig genome displayed in a bacteriophage lambda library. The provirus truncated in env due to cloning in lambda was complemented by PCR, and the recombinants were functionally characterized, confirming an increased infectivity in vitro compared to other PERV-C. Recombinant clone PERV-C(561) was chromosomally mapped by its 5'-proviral flanking sequences. Full-length PCR using 5'-and 3'-flanking primers specific to the PERV-C(561) locus verified that this specific SLAD/D haplotype pig harbors at least one full-length PERV-C provirus. The chromosomal location is different from that of the previously described PERV-C(1312) provirus, which was derived from the porcine cell-line MAX-T. The sequence data presented here provide further knowledge about PERV-C infectivity and contribute to targeted knockout in order to generate PERV-C-free founder animals. IMPORTANCE Yucatan SLAD/D haplotype miniature swine are candidates as organ donors for xenotransplantation. A full-length replication-competent PERV-C provirus was characterized. The provirus was chromosomally mapped in the pig genome. In vitro, the virus showed increased infectivity compared to other functional PERV-C isolates. Data may be used for targeted knockout to generate PERV-C free founder animals.

Transcriptomics and Selection Pressure Analysis Reveals the Influence Mechanism of PLIN1 Protein on the Development of Small Size in Min Pigs.

Body size is an important biological phenotypic trait that has attracted substantial attention. Small domestic pigs can serve as excellent animal models for biomedicine and also help meet sacrificial culture needs in human societies. Although the mechanisms underlying vertebral development regulating body size variation in domestic pigs during the embryonic period have been well described, few studies have examined the genetic basis of body size variation in post embryonic developmental stages. In this study, seven candidate genes-PLIN1, LIPE, PNPLA1, SCD, FABP5, KRT10 and IVL-significantly associated with body size were identified in Min pigs, on the basis of weighted gene co-expression network analysis (WGCNA), and most of their functions were found to be associated with lipid deposition. Six candidate genes except for IVL were found to have been subjected to purifying selection. PLIN1 had the lowest ω value (0.139) and showed heterogeneous selective pressure among domestic pig lineages with different body sizes (p < 0.05). These results suggested that PLIN1 is an important genetic factor regulating lipid deposition and consequently affecting body size variation in pigs. The culture of whole pig sacrifice in Manchu during the Qing Dynasty in China might have contributed to the strong artificial domestication and selection of Hebao pigs.

Transcriptomic analysis of testis and epididymis tissues from Banna mini-pig inbred line boars with single-molecule long-read sequencing†.

In mammals, testis and epididymis are critical components of the male reproductive system for androgen production, spermatogenesis, sperm transportation, as well as sperm maturation. Here, we report single-molecule real-time sequencing data from the testis and epididymis of the Banna mini-pig inbred line (BMI), a promising laboratory animal for medical research. We obtained high-quality full-length transcriptomes and identified 9879 isoforms and 8761 isoforms in the BMI testis and epididymis, respectively. Most of the isoforms we identified have novel exon structures that will greatly improve the annotation of testis- and epididymis-expressed genes in pigs. We also found that 3055 genes (over 50%) were shared between BMI testis and epididymis, indicating widespread expression profiles of genes related to reproduction. We characterized extensive alternative splicing events in BMI testis and epididymis and showed that 96 testis-expressed genes and 79 epididymis-expressed genes have more than six isoforms, revealing the complexity of alternative splicing. We accurately defined the transcribed isoforms in BMI testis and epididymis by combining Pacific Biotechnology Isoform-sequencing (PacBio Iso-Seq) and Illumina RNA Sequencing (RNA-seq) techniques. The refined annotation of some key genes governing male reproduction will facilitate further understanding of the molecular mechanisms underlying BMI male sterility. In addition, the high-confident identification of 548 and 669 long noncoding RNAs (lncRNAs) in these two tissues has established a candidate gene set for future functional investigations. Overall, our study provides new insights into the role of the testis and epididymis during BMI reproduction, paving the path for further studies on BMI male infertility.

Porcine IGF-1R synonymous mutations in the extracellular domain affect proliferation and differentiation of skeletal muscle cells.

OBJECTIVE: Miniature pigs are considered ideal organ donors for xenotransplantation in humans, but the mechanism underlying their dwarfism remains to be elucidated. IGF-1R is a crucial factor in body size formation in mammals, including skeletal muscle formation and development. The extracellular domain (ECD) binds to the ligand, a phenomenon that results in the activation of downstream pathways. METHODS: In this study, the coding sequences of two IGF-1R ECD haplotypes of the large Landrace (LP) pig and the small Bama Xiang (BM) pig were cloned into pcDNA3.1 vectors to generate pcDNA3.1-LP and pcDNA3.1-BM. The two recombinant vectors were then transfected into skeletal muscle cells. RESULTS: IGF-1R transcript was found to be expressed at higher levels in the pcDNA3.1-LP group than in the pcDNA3.1-BM group. The IGF-1R ECD from LP promoted cell proliferation and CyclinD1 expression, and promoted the phosphorylation of protein kinase B (to yield p-AKT). Moreover, the IGF-1R ECD from LP increased cell differentiation and the expression of myogenic determination factor (MyoD). CONCLUSION: Our data indicated that the IGF-1R ECD haplotypes between pig breeds with different body sizes affect IGF-1R expression, in turn affecting the proliferation and differentiation of skeletal muscle cells by activating downstream signalling pathways.

Comparative analysis of sperm DNA methylation supports evolutionary acquired epigenetic plasticity for organ speciation.

Aim: To perform a comparative epigenomic analysis of DNA methylation in spermatozoa from humans, mice, rats and mini-pigs. Materials & methods: Genome-wide DNA methylation analysis was used to compare the methylation profiles of orthologous CpG sites. Transcription profiles of early embryo development were analyzed to provide insight into the association between sperm methylation and gene expression programming. Results: We identified DNA methylation variation near genes related to the central nervous system and signal transduction. Gene expression dynamics at different time points of preimplantation stages were modestly associated with spermatozoal DNA methylation at the nearest promoters. Conclusion: Conserved genomic regions subject to epigenetic variation across different species were associated with specific organ functions, suggesting their potential contribution to organ speciation and long-term adaptation to the environment.

Infection Characteristics and Transcriptomics of African Swine Fever Virus in Bama Minipigs.

Animal experiments on African swine fever virus (ASFV) are vital to the study of ASFV; however, ASFV can only infect pigs, and animal experiments need to be performed in animal biosafety level 3 (ABSL-3) laboratories, meaning that many small ABSL-3 laboratories are unable to carry out in vivo ASFV experiments. Therefore, miniaturized experimental animals for ASFV infection are urgently needed. Here, we successfully isolated genotype II of ASFV SY-1 from wild boars and evaluated ASFV-infected Bama minipigs in a negative-pressure isolator of a small ABSL-3 laboratory. The pathological changes of ASFV-infected Bama minipigs were consistent with characteristic lesions of ASFV-infected domestic pigs and wild boars. All pigs died 5 to 14 days postinfection (dpi) through intramuscular injection. Viral genomic DNA from nasal, oral, and rectal swab samples was first detectable at 2 to 4 dpi. The common differentially expressed genes were clustered in the immune-related, metabolic, and inflammatory response pathways from the spleen and inguinal lymph node samples comparing infected to mock. In summary, these results demonstrated that the Bama minipig was an appropriate model for ASFV infection in small ABSL-3 laboratories that can accelerate the research of vaccines and antiviral drugs and uncover pathogenic mechanisms of ASFV infection. IMPORTANCE African swine fever virus (ASFV) can only infect pigs rather than other animals. However, the domestic pigs cannot be kept in small ABSL-3 laboratories for a long time due to the characteristics of rapid growth and large size, which hinder ASFV research, including research of vaccines, antiviral drugs, and mechanisms. In contrast, Bama minipigs have unique advantages consisting of low growth and small size. In the research, Bama minipigs were used to evaluate the characteristics of ASFV infection in small ABSL-3 laboratories. The pathological changes, viral shedding, and gene regulation were consistent with those of domestic pigs infected with ASFV. Therefore, Bama minipigs can be a suitable model for ASFV infection in small ABSL-3 laboratories.

Identification and validation of stable reference genes for quantitative real time PCR in different minipig tissues at developmental stages.

BACKGROUND: Quantitative real time PCR (qPCR) is a powerful tool to evaluate mRNA expression level. However, reliable qPCR results require normalization with validated reference gene(s). In this study, we investigated stable reference genes in seven tissues according to four developmental stages in minipigs. Six candidate reference genes and one target gene (ACE2) were selected and qPCR was performed. BestKeeper, geNorm, NormFinder, and delta Ct method through the RefFinder web-based tool were used to evaluate the stability of candidate reference genes. To verify the selected stable genes, relative expression of ACE2 was calculated and compared with each other. RESULTS: As a result, HPRT1 and 18S genes had lower SD value, while HMBS and GAPDH genes had higher SD value in all samples. Using statistical algorithms, HPRT1 was the most stable gene, followed by 18S, ß-actin, B2M, GAPDH, and HMBS. In intestine, all candidate reference genes exhibited similar patterns of ACE2 gene expression over time, whereas in liver, lung, and kidney, gene expression pattern normalized with stable reference genes differed from those normalized with less stable genes. When normalized with the most stable genes, the expression levels of ACE2 in minipigs highly increased in intestine and kidney at PND28, which is consistent with the ACE2 expression pattern in humans. CONCLUSIONS: We suggest that HPRT1 and 18S are good choices for analyzing all these samples across the seven tissues and four developmental stages. However, this study can be a reference literature for gene expression experiments using minipig because reference gene should be validated and chosen according to experimental conditions.