A REVERSE TRANSCRIPTION-POLYMERASE CHAIN-REACTION TECHNIQUE TO DETECTFELINE CYTOKINE GENES

The ability to detect feline cytokine expression would allow further c haracterization of the feline immune system, Bioassays are currently a vailable for the measurement of feline IL2, IL6 and TNF alpha but not for other biologically important cytokines. To detect the expression o f other cytokines, a reverse transcription-polymerase chain reaction ( RT-PCR) procedure was developed, Since feline cytokine gene sequences other than TNF alpha were not available, mammalian DNA and mRNA sequen ces for IL2, IFN gamma, IL4, IL6, IL10, IL12 and beta-actin, obtained from the Genbank database were compared and oligonucleotide primers ch osen from consensus sequences. To validate the cytokine and beta-actin primers, peripheral blood mononuclear cells from specific pathogen fr ee (SPF) cats were cultured in the presence of Con A for various perio ds of time (0-72 h). RNA was collected, reverse transcribed into cDNA, and the cDNA was amplified by PCR with each set of cytokine primer pa irs. RT-PCR products were hybridized with specific P-32 end-labeled in ternal oligonucleotide probes and then analyzed with the AMBIS imaging system to determine the kinetics of cytokine mRNA production. The bet a-actin signal was used to control for sample to sample variation in t he quantity of mRNA and variation in the RT and PCR reactions. Peak mR NA expression for most cytokines was found to occur between 2 to 4 h o f Con A stimulation. mRNA expression was correlated with cytokine bioa ctivity for IL2 and IL6. Peak IL2 bioactivity occurred after 8 h of Co n A stimulation, 4 h after the mRNA expression had peaked. Although IL 6 mRNA expression peaked between 2 and 4 h of stimulation, bioactivity was not detected until 8 h of stimulation and continued to increase o ver the next 24-48 h.