Analysis of gene expression patterns by microarray hybridization in blood mononuclear cells of SLA-DRB1 defined Canadian Yorkshire pigs
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The Swine Leukocyte Antigen (SLA) system encodes molecules for self-nonself discrimination and is associated with immune responses and disease resistance. Three lines of pigs defined by their SLA-DRB1 alleles were developed at the University of Guelph for xenotransplantation and immune response studies. The aim of this project was to explore the potential association between defined SLA-DRB1 alleles and gene transcriptional patterns of other immune-related genes in blood mononuclear cells.
Three SLA-DRB1 alleles were characterized using a RT-PCR-based sequencing method. The loci represented included a new allele, DRB1*04ns01. Next, microarray heterologous (bovine-porcine) hybridization together with qPCR were used to explore differential gene expression between SLA-DRB1-defined groups. Microarray analysis showed significant (p < 0.01) differential expression for 5 genes, mostly related to inflammation. Genes varied according to the comparison analyzed. Further testing with qPCR revealed the same trend of differential expression for 4 of the genes, although statistical significance was reached for only one.
A new SLA-DRB1 allele was characterized. A potential association was found between SLA-DRB1 alleles and inflammation-related genes. However, the influence of other genes cannot be ruled out. These preliminary findings agree with other studies linking MHC haplotypes and inflammation processes, including autoimmune disease. The study provides an initial view of the biological interactions between the SLA complex and other immune-related genes. Future studies will focus on characterization of SLA-haplotypes associated with these particular alleles and the dynamics of the immune response to antigenic challenges.
KeywordsProduction Trait Allele Group Swine Leukocyte Antigen Transcript Quantification Heterologous Hybridization
List of abbreviations
Lymphotoxin beta (TNF superfamily, member 3)
Bromodomain containing 2
Chemokine (C-C motif) ligand 4
Interleukin 1 beta
SLA class II DQ alpha
Toll-like receptor 2
Ribosomal protein L19, EBI IPD, European Bioinformatics Institute Immuno-Polymorphism Database
Basic Local Alignment Search Tool
Reverse transcription – polymerase chain reaction
Swine leukocyte antigen
Major histocompatibility complex
Blood mononuclear cells
Gene Expression Omnibus
Minimum information about a microarray experiment
Locally weighted scatter plot smoothing algorithm
False Discovery Rate.
The highly polymorphic MHC-encoded molecules are crucial for self-nonself discrimination in vertebrates. They constitute the major barrier for transplantation, contain numerous genes involved in immunological and non-immunological functions and are associated with resistance or susceptibility to various diseases. The two main classes, I and II, are involved in antigen presentation to T-cells. However, a large number of the genes in the MHC, like class III genes, are not directly related to this function [1, 2]. A total of 152 loci have been annotated within this region. In pigs, known as the SLA, the DRB genes show extensive polymorphism in exon 2 and the 135 available sequences identified to date are distributed into at least 10 confirmed allele groups .
Different SLA haplotypes have been associated with variation in immune response and disease, as well as reproduction and production traits . Therefore, SLA-defined pigs constitute an invaluable resource to study immune response, disease resistance and production traits, as well as an important large animal model for biomedical research [5, 6]. Three lines of commercial Yorkshire pigs with defined SLA-DRB1 genotypes were produced at the University of Guelph for xenotransplantation and immune response research [7, 8]. The aims of this study were to characterize the SLA-DRB1 alleles in these three pig lines and explore differential transcriptional activity between the three groups using heterologous (bovine probes – porcine targets) cDNA microarray and qPCR.
Animals and samples
Animal use was approved by the Animal Care Committee of the University of Guelph. Thirty-five pigs were included in the study (n = 6 for microarray analysis and n = 29 for qPCR). Pigs came from crossings of an outbred population selected for only by specific SLA-DRB1 alleles. Age of pigs ranged between 3–6 months and all pigs were in good general health at the time of sampling. Venous blood was collected in EDTA coated BD Vacutainer® collection tubes (BD – Canada, Oakville, ON, Canada) and processed immediately after collection. MNCs were isolated using Histopaque-1077 (Sigma-Aldrich Canada Ltd., Oakville, ON, Canada) and total RNA was extracted using TRIzol™ reagent (Invitrogen Canada Inc., Burlington, ON, Canada). Total RNA was treated with DNA-free (Ambion Inc., TX, USA) to eliminate genomic DNA contamination. Concentration and quality were assessed with an Agilent 2100 Bioanalyzer (Agilent Technologies Inc., Santa Clara, CA, USA).
SLA-DRB1 alleles characterization
cDNA Microarray experiments
Summary of results from cDNA microarray and qPCR data analysis
Comparison A: SLA-DRB1 alleles 0502/04ns01
Comparison B: SLA-DRB1 alleles 0502/0701
Comparison C: SLA-DRB1 alleles 0701/04ns01
Numerous associations have been established in swine between SLA haplotypes and features such as immune response and disease [16, 17], reproduction  and production traits . Many of these traits are not directly regulated by individual SLA genes but could rather be under the influence of non-classical MHC genes or controlled by downstream pathways yet to be described. The involvement of other closely linked genes, whose variants are in linkage disequilibrium (LD) can not be discarded [20, 21]. For example, it has been found that differential expression of LTB (also known as TNF beta) in MHC class II-defined B cell lines is associated with certain MHC class II haplotypes but not others. This association could be explained by LD between LTB and MHC haplotypes or by the influence of polymorphism in the MHC class II molecules and their interactions on the control of gene expression . Another example is represented by BRD2 in humans. This transcription factor, without an established immune function and located in the MHC class II region, is strongly linked to the MHC in most vertebrates .
Although it is not possible from the results in this study to establish a direct causal relationship between particular SLA-DRB1 alleles and differential transcription of inflammatory genes observed, it is undeniable that there seems to be an association. These observations will be better explained by the characterization of the haplotypes linked to these alleles and further exploration of the immune response in animals with defined MHC haplotypes.
Gene-specific primers and PCR conditions for relative quantification in the Light Cycler system
Primers (5'-> 3') b
Prod. size (bp) c
Ann. temp. (°C) d
Acq. temp. (°C) e
We wish to acknowledge the financial support of Ontario Pork Producers to BAM and the Government of Iran to RJJ. The assistance of the personnel at Arkell Research Station (University of Guelph), technical support of Sophia Lim, and assistance in statistical analysis of William Sears are greatly appreciated.
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