Effect of antiheparanase siRNA on DTH reactivity in vivo. Ears of oxazolone-sensitized BALB/c mice were electroporated with anti–mouse heparanase siRNA expression vector pSi2 (▪); empty vector pSUPER (▴); or received no plasmid or electroporation (♦), followed by challenge with the hapten 24 hours later. Hapten also was applied on the ears of 5 additional mice, which have not been previously sensitized or electroporated (▪). Three independent experiments were performed, and 5 mice per treatment were used. (A) Ear thickness was measured for 5 consecutive days after challenge. Inset. Effect of treatment with an inhibitor (ST1514) of heparanase enzymatic activity on DTH reactivity. ST1514 or vehicle alone was administered intraperitoneally prior to challenge and every hour thereafter (50 μg/injection) during the following 8 hours of the experiment. Filled bars: ear thickness in ST1514-treated mice; empty bars: ear thickness in vehicle-treated mice. (B) The ears in which DTH was induced following electroporation with pSi2 (left) or pSUPER (right) vectors were harvested 48 hours after challenge and processed for immunohistochemical analysis of heparanase expression (reddish staining; sebaceous glands are positively stained in all preparates, due to nonspecific absorption, as previously reported.³²  Top: magnification × 200; bottom: × 1000. Positively stained capillary endothelium is noted in the dermis of pSUPER but not pSi2-electroporated ear skin. (C) To demonstrate that electroporation ensures the actual delivery of plasmid DNA and its uniform expression in the ear tissue, the ears of male BALB/c mice were electroporated with a CMV-LUC construct, encoding for luciferase gene under the constitutive CMV promoter. Expression of luciferase in the mouse ears in vivo was monitored as described in “Materials and methods.”³²