Cloning bovine cytokine cDNA fragments and measuring bovine cytokine mRNA using the reverse transcription-polymerase chain reaction.
Bovine cytokine-specific primers and the reverse transcription-polymerase chain reaction (RT-PCR) were used to clone cDNA fragments that were specific for bovine IL-1 alpha, IL-1 beta, IL-2, and IFN-gamma. Specificity of the cDNA fragments was verified by sequence analysis based on known bovine IL-1 alpha, IL-1 beta, IL-2, and IFN-gamma gene sequences. In addition, RT-PCR was used to monitor cytokine mRNA expression in concanavalin A (Con A) and lipopolysaccharide (LPS)-stimulated bovine peripheral blood mononuclear cells (PBMC), and the results were compared with those obtained by measuring PBMC cytokine secretion using biologic assays. IL-1 activity in LPS-stimulated PBMC cultures was similar at 12 h and 24 h, although the activity decreased by approximately 40% at 48 h. IL-2 and IFN-gamma activity in supernatants of Con A-stimulated PBMC cultures was low at 12 h and reached maximum levels at 48 h. RT-PCR transcript analysis detected an increase in IL-1 alpha, IL-1 beta, IL-2, and IFN-gamma mRNA expression that was usually correlated with the detection of these soluble cytokines by the bioassays. These results indicate that RT-PCR is a sensitive and effective method of obtaining cDNA probes and that this technique can be used to monitor bovine cytokine mRNA expression.
Hutchinson
L E
LE
United States Department of Agriculture, Agriculture Research Service, Ames, IA 50010, USA.
Stevens
M G
MG
Olsen
S C
SC
D000818:Animals; D001483:Base Sequence; D002417:Cattle; D002478:Cells, Cultured; D003001:Cloning, Molecular; D003208:Concanavalin A; D016207:Cytokines / Q000096:biosynthesis / Q000235:genetics*; D017931:DNA Primers / Q000737:chemistry; D018076:DNA, Complementary / Q000235:genetics*; D005260:Female; D008070:Lipopolysaccharides; D008213:Lymphocyte Activation / Q000276:immunology; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000662:veterinary; D012313:RNA / Q000302:isolation & purification; D012333:RNA, Messenger / Q000032:analysis* / Q000096:biosynthesis*; D013601:T-Lymphocytes / Q000276:immunology; D014158:Transcription, Genetic
D016207:Cytokines; D017931:DNA Primers; D018076:DNA, Complementary; D008070:Lipopolysaccharides; D012333:RNA, Messenger; D003208:Concanavalin A; D012313:RNA