Development of a microarray for studying porcine cytokine production in blood mononuclear cells and intestinal biopsies.
A microarray for demonstration of a limited number of porcine cytokines was initiated. Polymerase chain reaction (PCR) products were synthesized for four house-keeping genes, cyclophilin, beta-actin, hypoxanthine phosphoribosyltransferase (HPRT) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and the following cytokines: interleukin (IL)-1beta, IL-4, IL-6, IL-8, IL-10, IL-12 p35, IL-12 p40, IL-18, interferon (IFN)-alpha, IFN-beta, IFN-gamma, tumour necrosis factor (TNF)-alpha, macrophage inhibition factor (MIF) and granulocyte/macrophage colony-stimulating factor (GM-CSF). Cytokine production was induced by incubation of porcine peripheral blood mononuclear cells (PBMC) with Concanavalin A (ConA) or oligodeoxynucleotide (ODN) 2216. RNA was isolated after 6 or 24 h from stimulated cells or unstimulated control cells and from intestinal biopsies. Cytokine expression was analysed using a 3-DNA Array 350(TM) labelling kit from Genisphere. Data were normalized using external control genes and analysed with the genepix pro 5.0 software. All the cytokines could be induced in PBMC and expressed on the array and the cytokines IL-6 and IFN-alpha were also analysed at protein level. All but one cytokine were expressed in samples from intestinal biopsies. Densitometric analyses of PCR products of the house-keeping genes were performed to validate the results from the microarray. Thus, this microarray will enable analyses of the cytokine profile during local and systemic infections in the pig.
Andersson
M
M
Department of Clinical Sciences, Section of Comparative Physiology and Medicine, Swedish University of Agricultural Sciences, Box 7018, SE 75007 Uppsala, Sweden. marit.andersson@kv.slu.se
Berg
M
M
Fossum
C
C
Jensen-Waern
M
M
D000818:Animals; D001483:Base Sequence; D003208:Concanavalin A / Q000494:pharmacology; D016207:Cytokines / Q000096:biosynthesis*; D005260:Female; D015870:Gene Expression; D007413:Intestinal Mucosa / Q000378:metabolism*; D007963:Leukocytes, Mononuclear / Q000276:immunology / Q000378:metabolism*; D008297:Male; D046228:Microarray Analysis / Q000662:veterinary*; D008969:Molecular Sequence Data; D009711:Nucleotides / Q000494:pharmacology; D016133:Polymerase Chain Reaction / Q000662:veterinary; D012333:RNA, Messenger / Q000032:analysis; D013552:Swine; D013997:Time Factors
D016207:Cytokines; D009711:Nucleotides; D012333:RNA, Messenger; D003208:Concanavalin A