A reverse transcription-polymerase chain reaction method to analyze porcine cytokine gene expression

被引:103
作者
Dozois, CM
Oswald, E
Gautier, N
Serthelon, JP
Fairbrother, JM
Oswald, IP
机构
[1] INRA, Lab Pharmacol Toxicol, F-31300 Toulouse, France
[2] ENVT, INRA, Lab Associe Physiopathol & Toxicol Expt, Toulouse, France
[3] ENVT, INRA, Lab Associe Microbiol Mol, Toulouse, France
[4] Univ Montreal, Fac Med Vet, GREMIP, Quebec City, PQ, Canada
关键词
cytokine; RT-PCR; porcine;
D O I
10.1016/S0165-2427(97)00039-1
中图分类号
R392 [医学免疫学]; Q939.91 [免疫学];
学科分类号
100102 ;
摘要
A reverse transcription-polymerase chain reaction (RT-PCR) method was developed in order to provide a highly sensitive, rapid, and simple means of simultaneously measuring the expression of porcine cytokines in immune cell populations. Oligonucleotide primers were designed to amplify porcine cytokine cDNA from genes encoding IL-1 alpha, IL-1 beta, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12, IFN-gamma, TNF-alpha, TNF-beta and the housekeeping genes beta-actin and cyclophilin by PCR. Primers were chosen from different exons to detect for possible genomic DNA contamination of samples. To validate RT-PCR, unstimulated and concanavalin A (ConA) stimulated porcine peripheral blood mononuclear cells (PBMCs) were cultured from 2 h to 72 h, RNA Inas extracted and reverse transcribed, and cDNA was amplified using the different I!rimer sets. Band intensities of PCR products were quantified by densitometric scanning and values were normalized against cyclophilin. For each of the cytokines, the kinetics of gene expression were similar among PBMCs isolated from different animals and could be grouped into two main patterns. Lymphocyte derived cytokines (IL-2, IL-4, IFN-gamma, and TNF-beta) exhibited low level expression in unstimulated cells and increased expression in ConA-stimulated PBMCs, IFN-gamma and IL-2 mRNA levels peaked at 24 h and returned to baseline by 72 h, whereas IL-4 and TNF-beta mRNA levels did not return to baseline by 72 h. Pn contrast, substantial mRNA levels for inflammatory cytokines (IL-1 alpha, IL-1 beta, IL-6, IL-8, IL-12, and TNF-alpha) and IL-10 were detected from both unstimulated and ConA-stimulated PBMCs. Results indicate that RT-PCR is a sensitive and convenient method to monitor cytokine mRNA expression In porcine samples. (C) 1997 Elsevier Science B.V.
引用
收藏
页码:287 / 300
页数:14
相关论文
共 29 条
  • [1] BAARSCH M, 1994, THESIS U MINNESOTA
  • [2] NUCLEOTIDE AND DEDUCED AMINO-ACID-SEQUENCE OF PORCINE INTERLEUKIN 4 CDNA DERIVED FROM LAMINA PROPRIA LYMPHOCYTES
    BAILEY, M
    PERRY, ACF
    BLAND, PW
    STOKES, CR
    HALL, L
    [J]. BIOCHIMICA ET BIOPHYSICA ACTA, 1993, 1171 (03) : 328 - 330
  • [3] BAILEY M, 1994, IMMUNOLOGY, V82, P577
  • [4] Cytokine mRNA levels in unmanipulated (ex vivo) and in vitro stimulated monkey PBMCs using a semi-quantitative RT-PCR and high sensitivity fluorescence-based detection strategy
    Benveniste, O
    Vaslin, B
    Villinger, F
    LeGrand, R
    Ansari, AA
    Dormont, D
    [J]. CYTOKINE, 1996, 8 (01) : 32 - 41
  • [5] MOLECULAR-IDENTIFICATION OF PORCINE INTERLEUKIN-10 - REGULATION OF EXPRESSION IN A KIDNEY ALLOGRAFT MODEL
    BLANCHO, G
    GIANELLO, P
    GERMANA, S
    BAETSCHER, M
    SACHS, DH
    LEGUERN, C
    [J]. PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 1995, 92 (07) : 2800 - 2804
  • [6] DETECTION AND QUANTITATION OF 5-HT1A AND 5-HT2A RECEPTOR MESSENGER-RNAS IN HUMAN HIPPOCAMPUS USING A REVERSE-TRANSCRIPTASE POLYMERASE CHAIN-REACTION (RT-PCR) TECHNIQUE AND THEIR CORRELATION WITH BINDING-SITE DENSITIES AND AGE
    BURNET, PWJ
    EASTWOOD, SL
    HARRISON, PJ
    [J]. NEUROSCIENCE LETTERS, 1994, 178 (01) : 85 - 89
  • [7] DETECTION OF CYTOKINE TRANSCRIPTIONAL PROFILES FROM BOVINE PERIPHERAL-BLOOD MONONUCLEAR-CELLS AND CD4(+) LYMPHOCYTES BY REVERSE-TRANSCRIPTASE POLYMERASE CHAIN-REACTION
    COVERT, J
    SPLITTER, G
    [J]. VETERINARY IMMUNOLOGY AND IMMUNOPATHOLOGY, 1995, 49 (1-2) : 39 - 50
  • [8] CDNA CLONING OF PORCINE INTERLEUKIN-2 BY POLYMERASE CHAIN-REACTION
    GOODALL, JC
    EMERY, DC
    BAILEY, M
    ENGLISH, LS
    HALL, L
    [J]. BIOCHIMICA ET BIOPHYSICA ACTA, 1991, 1089 (02) : 257 - 258
  • [9] MOLECULAR-CLONING OF PORCINE ALVEOLAR MACROPHAGE-DERIVED NEUTROPHIL CHEMOTACTIC FACTOR-I AND FACTOR-II - IDENTIFICATION OF PORCINE IL-8 AND ANOTHER INTERCRINE-ALPHA PROTEIN
    GOODMAN, RB
    FOSTER, DC
    MATHEWES, SL
    OSBORN, SG
    KUIJPER, JL
    FORSTROM, JW
    MARTIN, TR
    [J]. BIOCHEMISTRY, 1992, 31 (43) : 10483 - 10490
  • [10] CLONING, SEQUENCING AND REGULATION OF AN MESSENGER-RNA ENCODING PORCINE INTERLEUKIN-1-BETA
    HUETHER, MJ
    LIN, GF
    SMITH, DM
    MURTAUGH, MP
    MOLITOR, TW
    [J]. GENE, 1993, 129 (02) : 285 - 289