RADIATION HYBRID MAPPING OF ESTs FROM PORCINE PEYER'S PATCHES

¹ University of Minnesota, College of Veterinary Medicine, Department of Veterinary PathoBiology, 1988 Fitch Avenue, St. Paul, MN 55108 USA

The mucosal surface of the intestinal tract is the major interface with the environment. The gut absorbs nutrients under immunologic tolerance while selectively developing host defenses against enteric pathogens. Most antigen presentation occurs in Peyer’s patches, yet the details of immune events within the Peyer’s patches are just beginning to be delineated. Therefore, we generated a subtracted cDNA library from the jejunal Peyer’s patches harvested from healthy, 4-6 week old pigs. RNA was isolated immediately after tissue removal or after ex vivo stimulation by one of the following treatments: 1) no treatment, 2) Salmonella choleraesuis vaccine strain SC-54, 3) phorbol myristate acetate, concanavalin A, and 8-bromo-cyclic AMP, and 4) lipopolysaccharide and cholera toxin. Each treatment was performed in the presence and absence of cycloheximide. Purified polyA+ RNA was pooled from the treatment groups and used to make a stimulated cDNA library. Untreated tissue was used to create an unstimulated cDNA library in the same manner. The libraries were pooled and subtracted with porcine ST fibroblast RNA to create an enriched porcine Peyer’s patch library of approximately 6,000 clones with an average insert size of 1.0kbp. Since many of the ESTs obtained from this library were novel or not previously identified in pigs, the ESTs were placed on the genome map to improve comparative mapping efforts. Primers were designed and used to amplify across a 6500rad radiation hybrid panel (INRA Minnesota Porcine Radiation Hybrid Panel (IMpRH)). This work will assist in development of high density genetic maps for positional cloning of genes responsible for immune function in the gut.