The delivery volume with the highest mean luciferase expression was chosen as the optimal delivery condition for the immunization studies. or number of injections before response. A three injection series of the same plasmid DNA by particle bombardment elicited a significant rise in antibody titer in 3/5 rabbits. One rabbit developed antibody after 1 injection and two after 3 injections. In contrast, 0/5 rabbits receiving DNA by needle and syringe injection responded. In the jet injection and particle bombardment groups, gene expression levels in the skin did not predict response. While immune Gemcitabine responses were similar, luciferase gene expression levels in the skin following particle bombardment were 10C100 times higher than jet injection. Conclusion Low-volume jet injection is a simple, effective methodology for intradermal Rabbit Polyclonal to IL4 DNA immunization. Background Jet injection is a relatively simple and low-cost method for gene transfer into somatic tissues . Aside from the injector, no special equipment or solutions are required. Plasmid DNA is suspended at the desired concentration in one of several types of solution and injected into tissue. More than one plasmid construct can be injected simultaneously. Injection of antigen encoding plasmids can be combined with either adjuvants or plasmids encoding immune enhancing cytokines. Low-volume jet injection deliberately limits the volume of the injected solution and is utilized to target delivery to small tissue areas . In mice, multiple different routes of DNA immunization have been used successfully . Particle bombardment of skin is reported to be superior to both intra-dermal and intra-muscular needle and syringe injection for induction of immunity in mice when compared directly [4-8]. In Aotus monkeys and pigs, intra-dermal administration of DNA vaccine by needle and syringe is reported to be more effective at inducing an immune response than intra-muscular administration by needle and syringe [9,10]. High-volume jet injection (500 l) of either 100 or 1000 g DNA into ovine muscle or mammary gland [11,12] or 500 g DNA into four separate muscle sites in rabbits  induces humoral immunity. Multiple studies have demonstrated that particle bombardment of skin elicits immune responses in larger animals [14-18]. Similarly, high-volume jet injection (100 l) of either 50 or 250 g DNA into the skin of primates induces immunity . Delivery to the oral mucosa by jet injection elicited a higher IgA response than intranasal delivery in mice . However, none of these studies compared different Gemcitabine intracutaneous DNA delivery methods within the same study. This investigation tested the ability of low-volume jet injection to target delivery of a DNA vaccine to the skin for the purpose of inducing immunity. A direct comparison of the new methodology to the established techniques of particle bombardment and needle and syringe injection was performed. The experiments demonstrated that low-volume jet injection successfully induced immunity in rabbits. Moreover, there were no significant differences between jet injection and particle bombardment in the maximal antibody titers or number of injections before response. In contrast no rabbits receiving DNA vaccine by needle and syringe injection developed an antibody response. In the low-volume jet injection and particle bombardment groups, gene expression levels in the skin did not predict the magnitude of the immune response. While immune responses were comparable, luciferase gene expression levels in the skin following particle bombardment were 10C100 times higher than low-volume jet injection. Results and discussion Low-volume jet injection targets delivery of a plasmid Gemcitabine DNA solution to the epidermis and dermis of rabbits Injection path lengths were measured following low-volume jet injection of a 1% India ink solution. Injection paths averaged between 0.5 mm and 0.75 mm (Figure ?(Figure1).1). The injection paths did not extend through the dermis. These experiments show that low-volume jet injection specifically targeted delivery of a DNA solution to the skin and that the injection paths did not reach into the underlying tissue. Open in a separate window Figure 1 Penetration depth of low-volume jet injection into rabbit skin. The injection paths of three separate low-volume jet injections of a 1% India ink solution at 3 Bar into rabbit skin are illustrated in panels A (1 path) and B (2 paths). Penetration depths averaged between 0.5 mm and 0.75 mm. Arrows indicate injection points into the epidermis. Optimal Gemcitabine injection parameters for low-volume jet injection, particle bombardment and needle and syringe injection Optimal injection parameters for each technique were established by comparing relative mean expression levels of the luciferase reporter gene in the skin 48 hours after injection. Injection parameters that yielded the highest levels of luciferase expression were selected as optimal. Particle.