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Clinical and Diagnostic Laboratory Immunology, July 2004, p. 691-698, Vol. 11, No. 4
1071-412X/04/$08.00+0     DOI: 10.1128/CDLI.11.4.691-698.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Development of a Macroarray To Specifically Analyze Immunological Gene Expression in Swine

Terence N. Ledger,¹ Philippe Pinton,¹ Dorothée Bourges,² Patrick Roumi,³ Henri Salmon,² and Isabelle P. Oswald^1*

INRA, Laboratoire de Pharmacologie-Toxicologie, UR66,¹ Laboratoire des Xenobiotiques, UMR1089, 31931 Toulouse,³ Laboratoire de Pathologie Infectieuse et Immunologie, UR918, 37380 Nouzilly, France²

Received 9 February 2004/ Returned for modification 16 March 2004/ Accepted 6 April 2004

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.

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* Corresponding author. Mailing address: Laboratoire de Pharmacologie-Toxicologie, INRA, 180 Chemin de Tournefeuille, BP3, 31931 Toulouse Cedex 9, France. Phone: (33) 5 61 28 54 80. Fax: (33) 561285310. E-mail: ioswald{at}toulouse.inra.fr.

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Clinical and Diagnostic Laboratory Immunology, July 2004, p. 691-698, Vol. 11, No. 4
1071-412X/04/$08.00+0     DOI: 10.1128/CDLI.11.4.691-698.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.