Suppressive Subtraction Hybridization on Stimulated Primary Horse Macrophages

To study genes potentially involved in genetic resistance to infectious diseases in the horse, suppressive subtraction hybridization was used to identify genes expressed in primary horse macrophages after their stimulation with E. coli. Overnight culture of blood monocyte-derived macrophage cells was stimulated with E. coli K12 in ratio 40 E. coli cells to one macrophage cell. After 4 hours of incubation, non-phagocyted bacteria were washed away. Following next 20 hour incubation in MEM alpha containing 5 μg of gentamycin in 1 ml of media, mRNA was isolated and used in Clontech PCR-Select cDNA Subtraction Kit. Expression of several known horse genes, as well as some new ESTs (expressed sequence tags) showing sequence similarity with immunity-related genes from other species was identified