Microarray Expression Profiles of 20.000 Genes across 23 Healthy Porcine Tissues

BACKGROUND: Gene expression microarrays have been intensively applied to screen for genes involved in specific biological processes of interest such as diseases or responses to environmental stimuli. For mammalian species, cataloging of the global gene expression profiles in large tissue collections under normal conditions have been focusing on human and mouse genomes but is lacking for the pig genome. METHODOLOGY/PRINCIPAL FINDINGS: Here we present the results from a large-scale porcine study establishing microarray cDNA expression profiles of approximately 20.000 genes across 23 healthy tissues. As expected, a large portion of the genes show tissue specific expression in agreement with mappings to gene descriptions, Gene Ontology terms and KEGG pathways. Two-way hierarchical clustering identified expected tissue clusters in accordance with tissue type and a number of cDNA clusters having similar gene expression patterns across tissues. For one of these cDNA clusters, we demonstrate that possible tissue associated gene function can be inferred for previously uncharacterized genes based on their shared expression patterns with functionally annotated genes. We show that gene expression in common porcine tissues is similar to the expression in homologous tissues of human. CONCLUSIONS/SIGNIFICANCE: The results from this study constitute a valuable and publicly available resource of basic gene expression profiles in normal porcine tissues and will contribute to the identification and functional annotation of porcine genes

Gene expression profiles in testis of pigs with extreme high and low levels of androstenone

<p>Abstract</p> <p>Background:</p> <p>Boar taint is a major obstacle when using uncastrated male pigs for swine production. One of the main compounds causing this taint is androstenone, a pheromone produced in porcine testis. Here we use microarrays to study the expression of thousands of genes simultaneously in testis of high and low androstenone boars. The study allows identification of genes and pathways associated with elevated androstenone levels, which is essential for recognising potential molecular markers for breeding purposes.</p> <p>Results:</p> <p>Testicular tissue was collected from 60 boars, 30 with extreme high and 30 with extreme low levels of androstenone, from each of the two breeds Duroc and Norwegian Landrace. The samples were hybridised to porcine arrays containing 26,877 cDNA clones, detecting 563 and 160 genes that were differentially expressed (p < 0.01) in Duroc and Norwegian Landrace, respectively. Of these significantly up- and down-regulated clones, 72 were found to be common for the two breeds, suggesting the possibility of both general and breed specific mechanisms in regulation of, or response to androstenone levels in boars. Ten genes were chosen for verification of expression patterns by quantitative real competitive PCR and real-time PCR. As expected, our results point towards steroid hormone metabolism and biosynthesis as important biological processes for the androstenone levels, but other potential pathways were identified as well. Among these were oxidoreductase activity, ferric iron binding, iron ion binding and electron transport activities. Genes belonging to the cytochrome P450 and hydroxysteroid dehydrogenase families were highly up-regulated, in addition to several genes encoding different families of conjugation enzymes. Furthermore, a number of genes encoding transcription factors were found both up- and down-regulated. The high number of clones belonging to ferric iron and iron ion binding suggests an importance of these genes, and the association between these pathways and androstenone levels is not previously described.</p> <p>Conclusion:</p> <p>This study contributes to the understanding of the complex genetic system controlling and responding to androstenone levels in pig testis. The identification of new pathways and genes involved in the biosynthesis and metabolism of androstenone is an important first step towards finding molecular markers to reduce boar taint.</p

Tissue Remodelling following Resection of Porcine Liver

Aim. To study genes regulating the extracellular matrix (ECM) and investigate the tissue remodelling following liver resection in porcine. Methods. Four pigs with 60% partial hepatectomy- (PHx-) induced liver regeneration were studied over six weeks. Four pigs underwent sham surgery and another four pigs were used as controls of the normal liver growth. Liver biopsies were taken upon laparotomy, after three and six weeks. Gene expression profiles were obtained using porcine-specific oligonucleotide microarrays. Immunohistochemical staining was performed and a proliferative index was assessed. Results. More differentially expressed genes were associated with the regulation of ECM in the resection group compared to the sham and control groups. Secreted protein acidic and rich in cysteine (SPARC) and collagen 1, alpha 2 (COL1A2) were both upregulated in the early phase of liver regeneration, validated by immunopositive cells during the remodelling phase of liver regeneration. A broadened connective tissue was demonstrated by Masson’s Trichrome staining, and an immunohistochemical staining against pan-Cytokeratin (pan-CK) demonstrated a distinct pattern of migrating cells, followed by proliferating cell nuclear antigen (PCNA) positive nuclei. Conclusions. The present study demonstrates both a distinct pattern of PCNA positive nuclei and a deposition of ECM proteins in the remodelling phase of liver regeneration

Two-way hierarchical clustering of gene expression ratios.

<p>Heatmaps displays the log2(M) on a color scale from green indicating lower expression to red indicating a higher expression, interpolated over black for log2(M) = 0. A. Overview of entire hierarchical clustering showing 26.877 cDNAs (row-wise) and 23 tissues (column-wise). B. Enlarged view of selected cDNA cluster. BFE, Biceps femoris; CBE, Cerebellum; FAT, fat; FCO, Frontal cortex; HEA, heart; ISP, Infraspinatus; KID, kidney; LDO, Longissimus dorsi; LIN, large intestine; LIV, liver; LUN, lung; PAN, pancreas; PGL, pituitary gland; SIN, small intestine; SKI, skin; SME, Semimembranosus; SPL, spleen; SSP, Supraspinatus; STE, Semitendinosus; STO, stomach; TBR, Triceps brachii; THG, thyroid gland; VIN, Vastus intermedius.</p

Expression profiles of uncharacterized genes and co-expressed genes with known function.

<p>Each histogram represents the expression profiles of a single cDNA across all 23 tissues for two uncharacterized protein coding genes (PC_207218, PC_207224) and four co-expressed genes with known function (PC_207826: ABCA10, PC_214110: UBE2D2). Each bar represents the gene expression ratio (M) between the tissue sample and the common reference sample on the logarithmic scale. M values below zero, indicating lower gene expression level, are shown by green bars. M values above zero, indicating higher expression, are shown by red bars.</p

KEGG pathways for cDNAs representing differently expressed genes across tissues.

<p>Size of dots corresponds to the number of cDNAs (minimum = 10 and maximum = 253) that were tested to have a positive influence on the expression levels and color codes indicate tissue type. Only significant (P≤0.05) KEGG pathways represented by 50 or more cDNAs on the array were included.</p

Enriched GO BP terms for cDNAs representing positively regulated genes.

<p>Size of dots corresponds to the number of cDNAs that were tested (minimum = 6 and maximum = 1025) and color codes indicate tissue type. Only significant (P≤0.01) GO-BP terms represented by 50 or more cDNAs on the array were included.</p