Tomoko Eguchi-Ogawa

Biased distribution of single nucleotide polymorphisms (SNPs) in porcine Toll-like receptor 1 (TLR1), TLR2, TLR4, TLR5, and TLR6 genes

Toll-like receptors (TLRs) recognize various microbial components and induce immune responses. Polymorphisms in TLRs may influence their recognition of pathogen-derived molecules; swine TLRs are predicted to be associated with responses to infectious diseases such as pneumonia. In this study, we searched for single nucleotide polymorphisms (SNPs) in the coding sequences of porcine TLR1, TLR2, TLR4, TLR5, and TLR6 genes in 96 pigs from 11 breeds and elucidated 21, 11, 7, 13, and 11 SNPs, respectively, which caused amino acid substitutions in the respective TLRs. Distribution of these nonsynonymous SNPs was biased; many were located in the leucine-rich repeats, particularly in TLR1. These data demonstrated that the heterogeneity of TLR genes was preserved in various porcine breeds despite intensive breeding that was carried out for livestock improvement. It suggests that the heterogeneity in TLR genes is advantageous in increasing the possibility of survival in porcine populations.

PEDE (Pig EST Data Explorer) has been expanded into Pig Expression Data Explorer, including 10 147 porcine full-length cDNA sequences

We formerly released the porcine expressed sequence tag (EST) database Pig EST Data Explorer (PEDE; ), which comprised 68 076 high-quality ESTs obtained by using full-length-enriched cDNA libraries derived from seven tissues. We have added eight tissues and cell types to the EST analysis and have integrated 94 555 additional high-quality ESTs into the database. We also fully sequenced the inserts of 10 147 of the cDNA clones that had undergone EST analysis; the sequences and annotation of the cDNA clones were stored in the database. Further, we constructed an interface that can be used to perform various searches in the database. The PEDE database is the primary resource of expressed pig genes that are supported by full-length cDNA sequences. This resource not only enables us to pick cDNA clones of interest for a particular analysis, but it also confirms and thus contributes to the sequencing integrity of the pig genome, which is now being compiled by an international consortium (). PEDE has therefore evolved into what we now call ‘Pig Expression Data Explorer’.

Antibody Repertoire Development in Fetal and Neonatal Piglets. XI. The Relationship of Variable Heavy Chain Gene Usage and the Genomic Organization of the Variable Heavy Chain Locus

In this study, we have mapped the 3′ H chain V region (VH) genes and those in the H chain diversity, H chain joining, and 5′ portion of the H chain constant locus. We show that swine possess only two functional H chain diversity segments and only one functional H chain joining segment. These data help to explain more than a decade of observations on the preimmune repertoire of this species and reveal the vulnerability of swine to natural or designed mutational events. The results are consistent with earlier studies on the region containing Enh, Cμ, and Cδ while revealing that the ancestral IgG3 is the most 5′ Cγ gene. We also observed a recent duplication (∼1.6 million years ago) in the VH locus that contains six of the seven VH genes that comprise 75% of the preimmune repertoire. Because there are no known transfers of immune regulators or Ags that cross the placenta as in mice and humans, fetal VH usage must be intrinsically regulated. Therefore, we quantified VH usage in fetal piglets and demonstrated that usage is independent of the position of VH genes in the genome; the most 3′ functional VH gene (IGHV2) is rarely used, whereas certain upstream genes (IGHV14 and IGHV15) are predominately used early in fetal liver but seldom thereafter. Similar to previous studies, three VH genes account for 40% of the repertoire and six for ∼70%. This limited combinatorial diversity of the porcine VH repertoire further emphasizes the dependence on CDR3 diversity for generating the preimmune Ab repertoire of this species.

Influence of polymorphisms in porcine NOD2 on ligand recognition.

Nucleotide oligomerization domain 2 (NOD2) is a cytosolic pattern recognition receptor (PRR) that responds to muramyldipeptide (MDP), a component of peptidoglycans of gram positive and negative bacteria. NOD2 is involved in the modulation of signaling pathways for other PRRs, such as Toll-like receptors. Polymorphisms in NOD2 may evoke bowel disorders, and human Crohns disease is significantly correlated with mis-sense insertion of the NOD2 gene. Such polymorphisms affecting ligand recognition in the NOD2 gene may also influence bowel flora in livestock, which is compromised by bowel diseases such as diarrhea. We investigated the functional variance of mis-sense polymorphisms in ligand recognition by porcine NOD2. The 1949T>C polymorphism, located in the region encoding the hinge domain of the molecule, notably diminished the functional response of porcine NOD2 to MDP. By comparison, the 2197A>C polymorphism, localized in the region corresponding to leucine-rich repeats, significantly augmented the response of porcine NOD2 to the ligand. The 1949C allele was rare among pig breeds, suggesting that this mutation is a disadvantage to pigs in their immune response to microbes. The 2197C allele, in contrast, was widely distributed among Western breeds and is most likely to be derived from wild boars in Asia. This is the first report of a causal relationship between molecular function and polymorphisms in PRRs in non-primate, non-rodent mammals. These findings suggest that the 2197C allele might confer an immune response advantage in modern pig breeds and may be a useful marker for breeding aimed at disease resistance in pigs.

Genomic sequence encoding diversity segments of the pig TCR δ chain gene demonstrates productivity of highly diversified repertoire

To better understand the function and diversity of gammadelta T cells, we determined the genomic sequence encoding diversity (D) segments of the porcine TCR delta chain and its upstream regions, because pigs and other artiodactyls have relatively high proportions of gammadelta T cells. The revealed sequence contained 28 variable (V) alpha/delta segments, including 4 TRDV1 and at least 6 Ddelta segments, a much higher number than in humans and mice. All 6 of the Ddelta segments that had canonical recombination signal sequences were functionally utilized in expressed TCR delta chain genes. The multiplicity of Ddelta segments enabled the use of more than 3 Ddelta segments in a single functional TCR delta chain. The increased number of TCR delta segments was acquired by the duplication of the germline sequence, which occurred after the divergence of artiodactyls from primates and rodents. These data demonstrate that the pig is able to generate a highly diversified repertoire of TCR delta chain molecules.

Genomic structure of the whole D-J-C clusters and the upstream region coding V segments of the TRB locus in pig.

In the vertebrate immune system, T cells play a central role in host defense against microbial or viral infection. Previous studies suggested that at least two sets of TRBD-J-C clusters are harbored in the porcine genome. In this study, we determined 212,193 bp of a continuous porcine genomic sequence covering the entire TRBC region. EPHB6, TRPV6, TRY, and ten TRBV genes were conserved in the vicinity of the TRBD-J-C clusters. Interestingly, three TRBD-J-C clusters were identified in this sequence; each TRBD-J-C cluster consisted of one TRBD and seven TRBJ segments, with one TRBC region composed of four exons. The distribution of repetitive sequences and phylogenetic analysis indicated that the TRBD-J-C cluster, located at the center of the three clusters identified, had a structure combined with the others. Most of the TRBJ segments were available in public databases, suggesting that all three TRBD-J-C clusters are functional in pigs.

Large-scale sequencing based on full-length-enriched cDNA libraries in pigs: contribution to annotation of the pig genome draft sequence

BackgroundAlong with the draft sequencing of the pig genome, which has been completed by an international consortium, collection of the nucleotide sequences of genes expressed in various tissues and determination of entire cDNA sequences are necessary for investigations of gene function. The sequences of expressed genes are also useful for genome annotation, which is important for isolating the genes responsible for particular traits.ResultsWe performed a large-scale expressed sequence tag (EST) analysis in pigs by using 32 full-length-enriched cDNA libraries derived from 28 kinds of tissues and cells, including seven tissues (brain, cerebellum, colon, hypothalamus, inguinal lymph node, ovary, and spleen) derived from pigs that were cloned from a sow subjected to genome sequencing. We obtained more than 330,000 EST reads from the 5′-ends of the cDNA clones. Comparison with human and bovine gene catalogs revealed that the ESTs corresponded to at least 15,000 genes. cDNA clones representing contigs and singlets generated by assembly of the EST reads were subjected to full-length determination of inserts. We have finished sequencing 31,079 cDNA clones corresponding to more than 12,000 genes. Mapping of the sequences of these cDNA clones on the draft sequence of the pig genome has indicated that the clones are derived from about 15,000 independent loci on the pig genome.ConclusionsESTs and cDNA sequences derived from full-length-enriched libraries are valuable for annotation of the draft sequence of the pig genome. This information will also contribute to the exploration of promoter sequences on the genome and to molecular biology-based analyses in pigs.

Changes in gene expression in a porcine preadipocyte cell line during differentiation.

Adipocyte differentiation plays an important role in the formation of fat tissues in pigs and affects meat quality and productivity. Clarification of the nature of the pig genes that participate in adipocyte differentiation will provide a clue to the regulation of fat content and thickness in pig carcases by dietary control; it will also help to find target genes for exploring potentially useful polymorphisms for molecular breeding aimed at fat traits. We constructed a DNA oligomer microarray based on pig transcripts, and we used the array to investigate time-dependent changes in gene expression in the PSPA porcine preadipocyte cell line during differentiation into adipocytes. We selected genes with markedly altered expression (at least fivefold difference in comparison with expression in undifferentiated cells) and classified them into five groups according to gene expression pattern. In the early stage after stimulation of adipocyte differentiation, we observed up-regulation of many genes encoding proteins involved in regulating cell proliferation and transcription. Among the probes corresponding to transcripts that showed marked changes in expression, 27 were located within previously reported QTL regions for traits related to adipose tissues. These results will be valuable resources for finding the genes responsible for fat-related traits that have been identified in previous studies using various pig resource families.

Generation and characterization of RAG2 knockout pigs as animal model for severe combined immunodeficiency.

Pigs with severe combined immunodeficiency (SCID) are versatile animal models for human medical research because of their biological similarities to humans, suitable body size, and longevity for practical research. SCID pigs with defined mutation(s) can be an invaluable tool for research on porcine immunity. In this study, we produced RAG2-knockout pigs via somatic cell nuclear transfer and analyzed their phenotype. The V(D)J recombination processes were confirmed as being inactivated. They consistently lacked mature T and B cells but had substantial numbers of cells considered to be T- or B-cell progenitors as well as NK cells. They also lacked thymic medulla and lymphoid aggregations in the spleen, mesenteric lymph nodes, and ileal Peyers patches. We showed more severe immunological defects in the RAG2 and IL2RG double-knockout pig through this study. Thus, SCID pigs could be promising animal models not only for translational medical research but also for immunological studies of pigs themselves.

Cloning, Expression, and Polymorphisms of Natural Killer Cell Receptor NCR1 in Pigs

NCR1 (NKp46) is expressed on the surfaces of natural killer cells and recognizes hemagglutinin on the influenza virus. We cloned the NCR1 gene in pigs and found that porcine NCR1 was minimally expressed in the thymus, suggesting that NCR1 could be a useful marker of natural killer cells in pigs. We observed three nonsynonymous single nucleotide polymorphisms and one deletion of three nucleotides in the coding sequence of porcine NCR1; these may affect the function of NCR1. The polymorphisms detected here may be useful markers for breeding for influenza resistance in pigs.