A mixed-model approach for the analysis of cDNA microarray gene expression data from extreme-performing pigs after infection with Actinobacillus pleuropneumoniae

We proposed a novel statistical approach for the analysis of cDNA experiments based on mixed-model methodology combined with mixtures of distributions. Our objective was to detect genes that may be involved in conferring heritable differences in susceptibility to common infections in intensive pig production. We employed a microarray expression profiling strategy and a mixed-model approach to the analysis of the expression data. A cDNA microarray of pig with 6,420 probes from immune tissues and cells was used to compare gene expression in peripheral blood leukocytes of two pigs showing extreme performance in their response to infection with Actinobacillus pleuropneumoniae. Principal components analyses were used to identify the two most extreme-performing pigs after infection (i.e., pigs whose measured responses to infection fell at the extremes). Blood samples and expression profiles from 0 to 24 h after infection were compared using a bivariate, mixed-model approach, in which the effect gene x immunological status interaction was treated as a random effect. Bayesian model-based clustering via mixtures of normal distributions of the resulting BLUP of the random interaction was approached and resulted in a list of 307 differentially expressed genes, of which 179 were down-regulated in the susceptible pig. The majority of the differentially expressed genes were derived from a cDNA library of leukocytes of A. pleuropneumoniae-challenged pigs that were subtracted against leukocytes before the challenge. These results provide evidence that the proposed statistical approach was useful in enhancing the knowledge of the mechanisms involved in the genetics of the immune response.     

猪骨骼肌发育过程中免疫相关基因的表达分析

为探究猪骨骼肌发育过程中免疫相关基因的表达模式以及相关生物学功能,本研究根据蓝塘猪和长白猪骨骼肌10个发育时期的转录组数据,对各时期免疫相关基因的表达数目和表达量进行分析,鉴定了相邻发育时期之间以及同一时期品种间差异表达的免疫相关基因,并对后者进行功能注释分析。结果表明:在蓝塘猪与长白猪骨骼肌发育过程中存在1 129个表达的免疫相关基因;胚胎期35 d和49 d、胚胎期91 d和生后期2d这2组相邻时期之间的差异表达免疫相关基因数目最多;品种间差异表达免疫相关基因数目则在胚胎期49d以及生后期180d最多,且趋向于在蓝塘猪上调表达,并富集在NIK/NF-κB信号传导、细胞凋亡的负调控等生物学过程中。本研究表明免疫相关基因在猪骨骼肌发育过程中存在表达,长白猪与蓝塘猪骨骼肌生长发育差异可能与NF-κB介导的细胞凋亡有关。     

Experimental Staphylococcus aureus infection of the mammary gland induces region-specific changes in innate immune gene expression

Staphylococcus aureus is a prolific mastitis-causing bacterium that resides naturally in the environment of the dairy cow. The aim of this study was to profile immune gene expression in tissue from the alveolar, ductal, gland cistern and teat canal regions of the bovine mammary gland following intramammary infection with S. aureus. Quantitative real-time PCR (qPCR) was used to profile expression of innate immune genes including pattern recognition receptors (PRRs), cytokines, antimicrobial peptides (AMPs) and acute phase proteins (APPs). Consistent expression of Toll-like receptors (TLRs) 1-10 and NOD-like receptors (NODs) 1-2 was detected in all four tissue regions. Pro-inflammatory cytokines (IL6, IL17A and IL8) and anti-inflammatory cytokine (IL10) were induced in all 4 tissues. APP (SAA3 and HP) and AMP (DEFB4 and DEFB5) genes showed the greatest induction throughout the mammary gland in response to S. aureus, with particularly high expression in alveolar tissue (SAA3 and HP >133- and >80-fold respectively, P<0.05; DEFB4 and DEFB5 >9- and >27-fold respectively, P<0.05). Collectively, our data show both sentinel and effector immune functions throughout the mammary gland in response to S. aureus challenge.     

Patterns of gene expression in pig adipose tissue: insulin-like growth factor system proteins, neuropeptide Y (NPY), NPY receptors, neurotrophic factors and other secreted factors

Although cDNA microarray studies have examined gene expression in human and rodent adipose tissue, only one microarray study of adipose tissue from growing pigs has been reported. Total RNA was collected at slaughter from outer subcutaneous adipose tissue (OSQ) and middle subcutaneous adipose tissue (MSQ) from gilts at 90, 150, and 210 d (n=5 age(-1)). Dye labeled cDNA probes were hybridized to custom porcine microarrays (70-mer oligonucleotides). Gene expression of insulin-like growth factor binding proteins (IGFBPs), hormones, growth factors, neuropeptide Y (NPY) receptors (NPYRs) and other receptors in OSQ and MSQ changed little with age in growing pigs. Distinct patterns of relative gene expression were evident within NPYR and IGFBP family members in adipose tissue from growing pigs. Relative gene expression levels of NPY2R, NPY4R and angiopoietin 2 (ANG-2) distinguished OSQ and MSQ depots in growing pigs. We demonstrated, for the first time, the expression of IGFBP-7, IGFBP-5, NPY1R, NPY2R, NPY, connective tissue growth factor (CTGF), brain-derived neurotrophic factor (BDNF) and ciliary neurotrophic factor (CNTF) genes in pig adipose tissue with microarray and RT-PCR assays. Furthermore, adipose tissue CTGF gene expression was upregulated while NPY and NPY2R gene expression were significantly down regulated by age. These studies demonstrate that expression of neuropeptides and neurotrophic factors in pig adipose tissue may be involved in regulation of leptin secretion. Many other regulatory factors were not influenced by age in growing pigs but may be influenced by location or depot.     

乙型脑炎病毒感染PK15细胞的lncRNA差异表达谱分析

旨在分析乙型脑炎病毒(Japanese encephalitis virus,JEV)感染猪肾上皮细胞PK15后的lncRNA差异表达谱,探究宿主lncRNA在JEV感染和宿主防御中的潜在功能.本研究中利用JEV感染PK15细胞,感染36 h,收集细胞,同时设立未感染对照组,每组3个生物学重复.提取细胞总RNA,构建c...     

Effect of dietary phosphorus and its interaction with genetic background on global gene expression in porcine muscle

Environmental concerns and costs associated with dietary phosphorus (P) supplementation have lead to attempts to minimize the amount of P added to swine diets. In addition to its requirement for bone growth, dietary P is also necessary for muscular growth. To examine the effects of genetic background and dietary P on global gene expression in the muscle of young pigs, we utilized muscle tissue from 36 gilts sired from two different sire lines. These animals were fed either a P adequate, P deficient or P repletion diets for 14 days and showed differences in growth performance and bone integrity in response to the interaction of genetic background and dietary P. Total RNA from the loin muscle of these animals was obtained for microarray analysis. Significant differences (p < 0.01) in gene expression were seen based on the effect of sire line (339 genes), dietary P (18 genes) and the interaction between sire line and dietary P (31 genes). The microarray data were validated by semi-quantitative real-time PCR. These results support our hypothesis that genetic background and dietary P treatment can affect the homeorhetic control of P metabolism in pigs. Genes identified as differentially expressed in this study may be excellent candidate genes for additional work to elucidate genotype specific P requirements as well as to identify a genetic background that can maintain superior growth in a more environmentally friendly manner.     

日粮碳水化合物/蛋白质水平对240日龄不同品种猪肌内脂肪含量和胃肠道重量、长度及消化酶活性的影响

本试验旨在研究日粮碳水化合物/蛋白质水平对240日龄不同品种猪肌内脂肪(IMF)含量和胃肠道重量、长度及消化酶活性的影响,并探讨IMF含量与胃肠道重量、长度及消化酶活性的关系.试验采用2×2因子试验没计,即杜洛克×长白×大约克三元杂交猪(DLY)和太湖猪2个品种,每个品种分别饲喂高碳水化合物/低蛋白(HC/LP)和低碳水化合物/高蛋白(LC/HP)2种日粮.选用150日龄的DLY猪和太湖猪各12头,按体重相近、性别一致的原则共分为4个试验组,每个试验组6个重复,每个重复1头猪.饲养90 d后屠宰取样.结果表明:与LC/HP日粮相比,HC/LP日粮极显著提高了DLY猪的IMF含量(IMF为5.83%)(P<0.01),对太湖猪有提高的趋势(P>0.05),但2种日粮对2品种猪眼肌面积和背膘厚没有显著影响(P>0.05);HC/LP日粮显著(P<0.05)或极显著(P<0.01)降低了2品种猪的胰重和胰指数;无论在何种日粮下,太湖猪的胃、胰、小肠指数都极显著高于DLY猪(P<0.01);HC/LP日粮降低了2品种猪的消化酶活性,但对DLY猪的胰淀粉酶单位活性有所升高(P>0.05),其中对太湖猪的胃蛋白酶和胰脂肪酶活性的影响达到极显著水平(P<0.01).在LC/HP日粮下,太湖猪胰脂肪酶活性极显著高于DLY猪(P<0.01);在HC/LP日粮下,DLY猪的胰淀粉酶活性显著高于太湖猪(P<0.05).因此,HC/LP日粮在对2品种猪眼肌面积和背膘厚影响较小的情况下,可大幅度提高240日龄DLY猪的IMF含量,而大幅度降低了2品种猪的胰重和太湖猪的胃蛋白酶和胰脂肪酶活性,对2品种猪的胃、肝和小肠的重量或长度影响不大.     

Acute-phase protein response in pigs experimentally infected with Haemophilus parasuis

The acute-phase protein (APP) response to an infection caused by Haemophilus parasuis, the etiological agent of Glässer's disease in pigs, was characterized measuring serum concentrations of pig major acute-phase protein (pig MAP), haptoglobin (HPT), C-reactive protein (CRP) and apolipoprotein A-I (ApoA-I) in colostrum-deprived pigs. They were divided into six experimental groups: non-immunized control group (I); immunized with a non-commercial bacterin (II); with an OMP-vaccine (III); with a sublethal dose (IV); and with two commercial bacterins (V and VI). All groups were challenged intratracheally with 5 × 10⁹ CFU of H. parasuis 37 days after immunisation. The highest levels of the positive APPs (pig MAP, HPT and CRP) and the lowest levels of the negative APPs (ApoA-I) were observed in the animals that died as a consequence of the infection, both those in the non-inmunized and in the immunized groups. However, the surviving animals (all of them in groups II, V and VI, two pigs in group III, and three in group IV) showed a minor variation in APP response, mainly on day 1 post-challenge (p.c.), and then tended to recover the initial values. APP response was still less pronounced in the groups of pigs previously immunized with bacterins. In conclusion, APP response can reflect Glässer-disease ongoing, showing a correlation between the severity and duration of the clinical signs and lesions and the magnitude of changes in the APP levels.     

感染肺炎支原体姜曲海猪肺组织差异表达circRNAs的筛选与分析

为探明姜曲海猪感染猪肺炎支原体（Mycoplasma hyopneumoniae,Mhp）后肺组织环状RNA （circular RNA,circRNA）差异性表达谱及其在抗Mhp感染中的作用,试验以姜曲海猪为研究对象,分为感染组和对照组,人工感染Mhp 28 d后,解剖采集肺组织,采用高通量测序和生物信息学软件分析circRNA的表达情况。测序数据比对参考猪基因组序列,共鉴定到23 632个circRNAs,差异表达circRNAs为213个,其中97个上调,116个下调。随机选择4个差异表达circRNAs进行实时荧光定量PCR验证,检测结果与测序结果基本一致。差异表达circRNA来源基因可注释到包括抗原加工递呈、溶酶体、白细胞跨内皮迁移等免疫应答信号通路。circRNA-miRNA-mRNA靶标关系分析显示,筛选到海绵结合miRNA数量最多的3个差异表达circRNAs,分别为：circRNA-17284（21个）、circRNA-04848（19个）和circRNA-17270（19个）;预测到6个与免疫调控相关的靶向miRNAs,分别为：ssc-miR-4331、ssc-miR-370、ssc-miR-328、ssc-miR-30c-3p、ssc-miR-122和ssc-miR-125b。本研究测定了感染Mhp的猪肺组织circRNA表达谱,筛选到与免疫调控相关的差异表达circRNA,这有助于阐明姜曲海猪对Mhp的易感机制,为抗病育种研究提供了参考依据。     

Construction of a microarray specific to the chicken immune system: profiling gene expression in B cells after lipopolysaccharide stimulation

The objective of this study was to profile gene expression in cells of the chicken immune system. A low-density immune-specific microarray was constructed that contained genes with known functions in the chicken immune system, in addition to chicken-expressed sequence tags (ESTs) homologous with mammalian immune system genes, which were systematically characterized by bioinformatic analyses. Genes and ESTs that met the annotation criteria were amplified and placed on a microarray. The microarray contained 84 immune system gene elements. As a means of calibration, the microarray was then used to examine gene expression in chicken B cells after lipopolysaccharide stimulation. Differential gene expression was observed at 6, 12, and 24 h but not at 48 h after stimulation. The results were validated by semiquantitative polymerase chain reaction. The microarray showed a high degree of reproducibility, as demonstrated by intra- and interassay correlation coefficients of 0.97 and 0.95, respectively. Thus, the low-density microarray developed in this study may be used as a tool for monitoring gene expression in the chicken immune system.     

Cellular Localization,Expression Patterns of CCAR1 Gene,and Its Effect on Cell Proliferation in Pig

The aim of this study was to obtain the complete coding sequence (CDS) of CCAR1 gene, and to explore its subcellular localization, expression profile and its effects on cell prolification and action mechanism in pig. In this study, the cDNA from kidney tissue of 1-day-old Mashen pigs were used as the template to obtain the full-length CDS of CCAR1 gene by RT-PCR and sequencing. The cellular immunofluorescence staining was used to explore the subcellular localization of CCAR1 in PK15 cells. The temporal and spatial expression profile of CCAR1 was investigated by qRT-PCR in this study. The CCAR1 gene in PK15 cells was knocked out by using CRISPR/Cas9 gene editing technology, and the effects of CCAR1 gene on cell proliferation and expression of cell proliferation and apoptosis related genes were investigated by qRT-PCR, Western blot and CCK8 (cell counting kit 8) technologies in this experiment. The results showed that the complete CDS region of pig CCAR1 gene was 3 459 bp in length (MH301308.1). CCAR1 protein was localized in both cytoplasm and nucleus of PK15 cells. The expression profiles of CCAR1 mRNA between Large White and Mashen pigs was similar, which was expressed in all detected tissues, with the highest expression in kidney and small intestine, middle expression in spleen, liver, cerebellum and muscle, and the lowest expression in heart and subcutaneous fat. Temporal expression results showed that CCAR1 was expressed in both psoas muscle and longissimus dorsi muscle at 3 developmental stages both in Mashen and Large White pigs. The CRISPR/Cas9 gene editing system effectively reduced the expression of CCAR1. CCK8 results showed that after 48 hours of transfection, compared with the control group, the proliferation of cells in the experimental groups were extremely significantly inhibited (P<0.01). After CCAR1 gene knocked out, the expression level of Mki67, a marker of cell proliferation, was significantly decreased (P<0.05). There was no significant difference in the expression level of Caspase3 and the core protein β-catenin in Wnt pathway between control group and experimental groups, and the expression level of downstream target gene C-myc of Wnt pathway was decreased significantly(P<0.05). CCAR1 gene was expressed almost in all tissues at different developmental stages, and affected cell proliferation by regulating the expression levels of Mki67 and C-myc, and played an important role in growth and development of pig.