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Development of a macroarray to specifically analyze immunological gene expression in swine.

ABSTRACT: DNA arrays are useful tools for simultaneously studying the expressions of a large number of genes. Herein, we describe the construction and the optimization of conditions for a low-density DNA macroarray specific for the porcine immune system. This specific DNA macroarray contains 63 gene products, including 20 cytokines, 11 chemokines, and 12 immunologically relevant receptors. It was constructed by designing gene-specific oligonucleotide primers from porcine sequences available in the EMBL or TIGR expressed sequence tag data bank and using primers from conserved regions of aligned sequences from other species for sequences unavailable for swine. Amplicons produced by reverse transcription-PCR were cloned, sequenced, and spotted onto nylon filters. A trial DNA array was first produced to optimize the intensity, specificity, and variability of signals from amplicons amplified with either gene-specific or universal primers. The DNA macroarray was then validated by comparing the gene expression profile of nonstimulated peripheral blood mononuclear cells (PBMCs) to that of phorbol 12-myristate 13-acetate and ionomycin (PMA-Iono)-stimulated PBMCs from three different animals over a 48-h time period. As already described for more conventional techniques, we showed that certain genes, such as those for CD40, gamma interferon, interleukin 2 (IL-2), the IL-2 receptor, and tumor necrosis factor alpha, were upregulated in PMA-Iono-stimulated PBMCs. A detailed analysis also indicated a downregulation of several genes which are expressed mainly by macrophages (IL-1, IL-8, AMCF-1, natural-resistance-associated macrophage protein, neutrophil chemotactic protein, DAP-12, and monocyte chemoattractant protein) in samples stimulated for 24 h with PMA-Iono compared to their levels of expression in control samples. These results indicate that the DNA macroarray that we constructed can be a useful tool for simultaneously monitoring the mRNA expression of immunologically relevant genes in different porcine samples.

SUBMITTER: Ledger TN

PROVIDER: S-EPMC440605 | biostudies-literature | 2004 Jul

REPOSITORIES: biostudies-literature

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Development of a macroarray to specifically analyze immunological gene expression in swine.

Ledger Terence N TN Pinton Philippe P Bourges Dorothée D Roumi Patrick P Salmon Henri H Oswald Isabelle P IP

Clinical and diagnostic laboratory immunology 20040701 4

DNA arrays are useful tools for simultaneously studying the expressions of a large number of genes. Herein, we describe the construction and the optimization of conditions for a low-density DNA macroarray specific for the porcine immune system. This specific DNA macroarray contains 63 gene products, including 20 cytokines, 11 chemokines, and 12 immunologically relevant receptors. It was constructed by designing gene-specific oligonucleotide primers from porcine sequences available in the EMBL or ...[more]

PMID: 15242943

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Project description:BACKGROUND: In higher plants, sugars are not only nutrients but also important signal molecules. They are distributed through the plant via sugar transporters, which are involved not only in sugar long-distance transport via the loading and the unloading of the conducting complex, but also in sugar allocation into source and sink cells. The availability of the recently released grapevine genome sequence offers the opportunity to identify sucrose and monosaccharide transporter gene families in a woody species and to compare them with those of the herbaceous Arabidopsis thaliana using a phylogenetic analysis. RESULTS: In grapevine, one of the most economically important fruit crop in the world, it appeared that sucrose and monosaccharide transporter genes are present in 4 and 59 loci, respectively and that the monosaccharide transporter family can be divided into 7 subfamilies. Phylogenetic analysis of protein sequences has indicated that orthologs exist between Vitis and Arabidospis. A search for cis-regulatory elements in the promoter sequences of the most characterized transporter gene families (sucrose, hexoses and polyols transporters), has revealed that some of them might probably be regulated by sugars. To profile several genes simultaneously, we created a macroarray bearing cDNA fragments specific to 20 sugar transporter genes. This macroarray analysis has revealed that two hexose (VvHT1, VvHT3), one polyol (VvPMT5) and one sucrose (VvSUC27) transporter genes, are highly expressed in most vegetative organs. The expression of one hexose transporter (VvHT2) and two tonoplastic monosaccharide transporter (VvTMT1, VvTMT2) genes are regulated during berry development. Finally, three putative hexose transporter genes show a preferential organ specificity being highly expressed in seeds (VvHT3, VvHT5), in roots (VvHT2) or in mature leaves (VvHT5). CONCLUSIONS: This study provides an exhaustive survey of sugar transporter genes in Vitis vinifera and revealed that sugar transporter gene families in this woody plant are strongly comparable to those of herbaceous species. Dedicated macroarrays have provided a Vitis sugar transporter genes expression profiling, which will likely contribute to understand their physiological functions in plant and berry development. The present results might also have a significant impact on our knowledge on plant sugar transporters.

Dataset Information

Development of a macroarray to specifically analyze immunological gene expression in swine.

Publications

Development of a macroarray to specifically analyze immunological gene expression in swine.

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