Project description:Domestication of pig results in modifications of many traits, including fatness traits, which are important in pig production since they have effect on meat quality, fattening efficiency, reproduction and immunity.In this study, we investigate 3D genome organization and transcriptomic characterization of adipose tissues (ATs) between wild boars and Bama pig, a typical indigenous domestic pig in China, to uncover molecular mechanisms of fatness-phenotypic shifts.
Project description:For gaining additional insights into the composition of the testicular proteome of the domestic pig (Sus scrofa domestica), we conducted 2DE-MS. Two-dimensional SDS PAGE was run on testicular lysates of three boars, with three gels per boar. Upon matching across gels, we arbitrarily selected protein spots for mass spectrometry analysis. Excised slices were vacuum dried and soaked with digestion buffer containing trypsin (0.01 μg/μl), followed by overnight incubation at 37°C in the same buffer without trypsin. Subsequently, peptides were extracted in solvents of increasing acetonitrile content, by sonication. Upon vacuum-centrifugation, peptides were reconstituted in 0.1% formic acid (FA). Following this, peptides were fractionated by reversed phase liquid chromatography (C18; buffer A: 0.1% FA dissolved in HPLC-H2O; buffer B: 0.1% FA, dissolved in CAN; flow-rate: 0.4 µL/min; gradient: 2-30% in 30 minutes). Eluted peptides were injected via an electrospray ionization interface into a Q-TOF mass spectrometer (one boar, Q TOF Ultima, Micromass/Waters, Manchester, UK) and an ion-trap mass spectrometer (two other boars, XCT ion-trap, Agilent Technologies, Waldbronn, Germany). We used ProteomeDiscoverer 2.4 (Thermo Fisher Scientific, San Jose, USA) for peptide and protein identification. Using Sequest HT, we searched peak lists (*.mgf) against the Sus scrofa reference proteome database (UniProt Proteome ID: UP000008227, 49,793 proteins).
Project description:The domestic pig (Sus scrofa) provides a large animal model for human innate immune responses and inflammation that is also economically important in its own right. Results: We demonstrate that macrophages can be harvested from 3 different compartments of the pig (lungs, blood and bone-marrow), cryopreserved and subsequently recovered and differentiated in CSF-1. We have performed surface marker analysis and gene expression profiling on macrophages from these compartments, comparing 25 animals from 5 different breeds and their response to lipopolysaccharide. The results provide a clear distinction between alveolar macrophages (AM) and monocyte-derived (MDM) and bone-marrow-derived macrophages (BMDM). In particular, the lung macrophages express the growth factor, Flt1 and its ligand, VEGFA at high levels, suggesting a distinct pathway of growth regulation. We confirm that pig macrophages more closely resemble human, than mouse, in their set of LPS-inducible genes. Relatively few genes showed breed-specific differential expression, notably CXCR2 and CD302 in alveolar macrophages. In contrast, there was substantial inter-individual variation between pigs within breeds, mostly affecting genes annotated as being involved in immune responses. Conclusions: Future research will address whether such variation is heritable, and might form the basis for selective breeding for disease resistance or functional genomics. The domestic pig (Sus scrofa) provides a large animal model for human innate immune responses and inflammation that is also economically important in its own right. We demonstrate that macrophages can be harvested from 3 different compartments of the pig (lungs, blood and bone-marrow), cryopreserved and subsequently recovered and differentiated in CSF-1. We have performed surface marker analysis and gene expression profiling on macrophages from these compartments, comparing 25 animals from 5 different breeds and their response to lipopolysaccharide. The results provide a clear distinction between alveolar macrophages (AM) and monocyte-derived (MDM) and bone-marrow-derived macrophages (BMDM). In particular, the lung macrophages express the growth factor, Flt1 and its ligand, VEGFA at high levels, suggesting a distinct pathway of growth regulation. We confirm that pig macrophages more closely resemble human, than mouse, in their set of LPS-inducible genes. Relatively few genes showed breed-specific differential expression, notably CXCR2 and CD302 in alveolar macrophages. In contrast, there was substantial inter-individual variation between pigs within breeds, mostly affecting genes annotated as being involved in immune responses. Future research will address whether such variation is heritable, and might form the basis for selective breeding for disease resistance or functional genomics. 140 Affymetrix Snowball microarray were analysed from 5 different breed (DR, LR, LW, PIE and HAM). 5 pig per breed were used and cells were harvested from Lungs, blood and bone-marrow (AM, MDM and BMDM). Cells were left untreated (0h) or stimulated with LPS Salmonella enterica serotype minnesota Re 595 - 100ng/ml (7h)