A reverse transcription-polymerase chain reaction technique to detect feline cytokine genes.
The ability to detect feline cytokine expression would allow further characterization of the feline immune system. Bioassays are currently available for the measurement of feline IL2, IL6 and TNF alpha but not for other biologically important cytokines. To detect the expression of 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 sequences for IL2, IFN gamma, IL4, IL6, IL10, IL12 and beta-actin, obtained from the Genbank database were compared and oligonucleotide primers chosen from consensus sequences. To validate the cytokine and beta-actin primers, peripheral blood mononuclear cells from specific pathogen free (SPF) cats were cultured in the presence of Con A for various periods 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 pairs. RT-PCR products were hybridized with specific 32P end-labeled internal oligonucleotide probes and then analyzed with the AMBIS imaging system to determine the kinetics of cytokine mRNA production. The beta-actin signal was used to control for sample to sample variation in the quantity of mRNA and variation in the RT and PCR reactions. Peak mRNA expression for most cytokines was found to occur between 2 to 4 h of Con A stimulation. mRNA expression was correlated with cytokine bioactivity for IL2 and IL6. Peak IL2 bioactivity occurred after 8 h of Con A stimulation, 4 h after the mRNA expression had peaked. Although IL6 mRNA expression peaked between 2 and 4 h of stimulation, bioactivity was not detected until 8 h of stimulation and continued to increase over the next 24-48 h.
Rottman
J B
JB
Department of Microbiology, Parasitology and Pathology, College of Veterinary Medicine, North Carolina State University, Raleigh 27606, USA.
Freeman
E B
EB
Tonkonogy
S
S
Tompkins
M B
MB
D000818:Animals; D001483:Base Sequence; D015139:Blotting, Southern / Q000662:veterinary; D002415:Cats / Q000235:genetics*; D002478:Cells, Cultured; D016384:Consensus Sequence; D016207:Cytokines / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics*; D017931:DNA Primers / Q000737:chemistry; D004587:Electrophoresis, Agar Gel / Q000662:veterinary; D015870:Gene Expression; D008213:Lymphocyte Activation; D008214:Lymphocytes / Q000737:chemistry / Q000378:metabolism; D008297:Male; D008969:Molecular Sequence Data; D015345:Oligonucleotide Probes / Q000737:chemistry; D016133:Polymerase Chain Reaction / Q000379:methods / Q000662:veterinary*; D012333:RNA, Messenger / Q000096:biosynthesis / Q000302:isolation & purification; D012680:Sensitivity and Specificity; D013047:Specific Pathogen-Free Organisms; D014158:Transcription, Genetic
D016207:Cytokines; D017931:DNA Primers; D015345:Oligonucleotide Probes; D012333:RNA, Messenger