CONSTRUCTION OF INTERNAL CDNA COMPETITORS FOR MEASURING IL-10 AND IL-12 CYTOKINE GENE-EXPRESSION IN SWINE

A competitive PCR assay (cPCR) was used to quantify swine cytokine res ponses to parasite infection. Internal standards (deleted cDNA competi tor molecules [DcDNA mimics]) were produced and tested for swine inter leukin-12 (IL-12), interleukin-10 (IL-10) and hypoxanthine phosphoribo syltransferase (HPRT) from PCR generated cDNA cloned in plasmid vector s. Deletion clones for the cDNA competitor molecules (DcDNA mimics) we re generated for IL-10, IL-12 and HPRT by PCR in a single step and ver ified by (1) amplification of the expected smaller PCR product with th e original primers, (2) appropriate fragment size released by restrict ion digestion of the deleted clone, and (3) correct sequence of the ne w DcDNA insert. DcDNA mimics were used to quantitate cytokine gene mRN A production during experimental and natural infections of swine with the gastrointestinal nematode parasite Trichuris suis. Mesenteric lymp h node cells were collected from control and infected pigs at the time of maximal pathogenicity (35 days after infection) and snap frozen. A fter RNA extraction, samples were reverse transcribed (RT) to cDNA. cP CR was performed using the housekeeping gene HPRT DcDNA mimic and HPRT specific primers to insure RNA integrity and concentration. Cytokine cDNA content in these samples was then quantitated using cytokine mimi cs and gene specific primers. IL-10 gene expression in MLN draining th e colon of pigs experimentally infected with T. suis increased 10-20 f old at day 35 compared to control pigs. IL-12 gene expression was not detectable in MLN of these pigs, but was detectable in MLN of pigs exp osed naturally to T. suis on a contaminated dirt lot that also exhibit ed signs of secondary bacterial invasion. Swine IL-10 and IL-12 gene e xpression can be quantitated in local mesenteric tissues. This cPCR as say will enable scientists to quantitate cytokine gene expression in s wine and determine the nature of immune responses to important infecti ous diseases. (C) 1998 Published by Elsevier Science B.V. All rights r esented.