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Analysis of gene expression patterns by microarray hybridization in blood mononuclear cells of SLA-DRB1 defined Canadian Yorkshire pigs.

Nino-Soto MI, Jozani RJ, Bridle B, Mallard BA - BMC Res Notes (2008)

Bottom Line: Microarray analysis showed significant (p < 0.01) differential expression for 5 genes, mostly related to inflammation.A potential association was found between SLA-DRB1 alleles and inflammation-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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Pathobiology, Ontario Veterinary College, University of Guelph, Guelph, Ont, N1G 2W1, Canada. mnino@uoguelph.ca

ABSTRACT

Background: 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.

Findings: 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.

Conclusion: 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.

No MeSH data available.


Related in: MedlinePlus

Nucleotide and protein sequence alignment of SLA-DRB1 alleles. Multiple sequence alignment for (a) nucleotide and (b) protein of published SLA-DRB1*0403 alleles (designated by their GenBank accession numbers) and *04ns01 (new allele). The black arrows mark (a) the point mutation and (b) the corresponding amino acid residue substitution.
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Figure 1: Nucleotide and protein sequence alignment of SLA-DRB1 alleles. Multiple sequence alignment for (a) nucleotide and (b) protein of published SLA-DRB1*0403 alleles (designated by their GenBank accession numbers) and *04ns01 (new allele). The black arrows mark (a) the point mutation and (b) the corresponding amino acid residue substitution.

Mentions: The SLA-DRB1 alleles were characterized according to Ho et al. (2006). Briefly, total RNA was reverse transcribed with the ThermoScript™ RT-PCR system using oligo d(T) primers (Invitrogen). A specific SLA-DRB1 coding region was amplified with PfuUltra™ Hotstart High-Fidelity DNA Polymerase (Stratagene, La Jolla, CA, USA) using a final concentration of 3 mM of MgCl2 and annealing temperature at 55°C. PCR products were gel-extracted with the QIAquick Gel Extraction Kit (Qiagen, Mississauga, ON, Canada) and purified PCR products were cloned using the Zero Blunt® TOPO® PCR cloning kit (Invitrogen). At least 7 colonies per animals were sent for sequencing. Each allele was characterized by sequencing using both forward and reverse primers, from at least two pigs per litter. The complete coding sequence was obtained by overlapping the forward and reverse fragments. Comparison with currently available sequences (GenBank and EBI-IPD-MHC, SLA section databases) was performed using BLAST [9] and ClustalW [10]. Two lines carried the SLA-DRB1*0502 and SLA-DRB1*0701 alleles respectively (Smith et al, 2005) as determined by a 100% homology between the sequences obtained from test samples and the corresponding published sequences. The third line had a novel allele, differing in one bp at position +118 in exon 2 with SLA-DRB1*0403. This difference corresponds to a point mutation substituting a cytosine for a guanine, which translates in the substitution of an arginine for a glycine residue in the protein encoded by this new allele (Figure 1). Sequences were submitted to GenBank [Genbank: EU087426, EU087427 and EU087428] and the EBI IPD-MHC (SLA section) which resulted in the assignment of the SLA-DRB1*04ns01 provisional name to the new allele, approved by the MHC Nomenclature Committee.


Analysis of gene expression patterns by microarray hybridization in blood mononuclear cells of SLA-DRB1 defined Canadian Yorkshire pigs.

Nino-Soto MI, Jozani RJ, Bridle B, Mallard BA - BMC Res Notes (2008)

Nucleotide and protein sequence alignment of SLA-DRB1 alleles. Multiple sequence alignment for (a) nucleotide and (b) protein of published SLA-DRB1*0403 alleles (designated by their GenBank accession numbers) and *04ns01 (new allele). The black arrows mark (a) the point mutation and (b) the corresponding amino acid residue substitution.
© Copyright Policy - open-access
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2529311&req=5

Figure 1: Nucleotide and protein sequence alignment of SLA-DRB1 alleles. Multiple sequence alignment for (a) nucleotide and (b) protein of published SLA-DRB1*0403 alleles (designated by their GenBank accession numbers) and *04ns01 (new allele). The black arrows mark (a) the point mutation and (b) the corresponding amino acid residue substitution.
Mentions: The SLA-DRB1 alleles were characterized according to Ho et al. (2006). Briefly, total RNA was reverse transcribed with the ThermoScript™ RT-PCR system using oligo d(T) primers (Invitrogen). A specific SLA-DRB1 coding region was amplified with PfuUltra™ Hotstart High-Fidelity DNA Polymerase (Stratagene, La Jolla, CA, USA) using a final concentration of 3 mM of MgCl2 and annealing temperature at 55°C. PCR products were gel-extracted with the QIAquick Gel Extraction Kit (Qiagen, Mississauga, ON, Canada) and purified PCR products were cloned using the Zero Blunt® TOPO® PCR cloning kit (Invitrogen). At least 7 colonies per animals were sent for sequencing. Each allele was characterized by sequencing using both forward and reverse primers, from at least two pigs per litter. The complete coding sequence was obtained by overlapping the forward and reverse fragments. Comparison with currently available sequences (GenBank and EBI-IPD-MHC, SLA section databases) was performed using BLAST [9] and ClustalW [10]. Two lines carried the SLA-DRB1*0502 and SLA-DRB1*0701 alleles respectively (Smith et al, 2005) as determined by a 100% homology between the sequences obtained from test samples and the corresponding published sequences. The third line had a novel allele, differing in one bp at position +118 in exon 2 with SLA-DRB1*0403. This difference corresponds to a point mutation substituting a cytosine for a guanine, which translates in the substitution of an arginine for a glycine residue in the protein encoded by this new allele (Figure 1). Sequences were submitted to GenBank [Genbank: EU087426, EU087427 and EU087428] and the EBI IPD-MHC (SLA section) which resulted in the assignment of the SLA-DRB1*04ns01 provisional name to the new allele, approved by the MHC Nomenclature Committee.

Bottom Line: Microarray analysis showed significant (p < 0.01) differential expression for 5 genes, mostly related to inflammation.A potential association was found between SLA-DRB1 alleles and inflammation-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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Pathobiology, Ontario Veterinary College, University of Guelph, Guelph, Ont, N1G 2W1, Canada. mnino@uoguelph.ca

ABSTRACT

Background: 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.

Findings: 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.

Conclusion: 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.

No MeSH data available.


Related in: MedlinePlus