to B cells and induced cytokine production from these cells. More recently, HRF was shown to stimulate bronchial epithelial cells to produce IL-8 and GM-CSF [18]. Thus, HRF, in addition to functioning as a histamine releasing factor, can modulate secretion of cytokines from human basophils, eosinophils, T cells, B cells and epithelial cells, firmly establishing HRF’s extracellular role. The importance of the association of HRF with human allergic disease has been previously documented in numerous publications. It should be noted that while these observations were made with crude HRF, the same exists with recombinant material. For instance, HRF has been found in human respiratory secretions (BAL) and skin blister fluids [1]. Furthermore, sensitivity to HRF was restricted to a subpopulation of atopic individuals [19]. In a separate study of ragweed allergic patients, there was a significant correlation between the intensity of symptoms in the late phase reaction and basophil histamine release to HRF [20]. In a third study, only basophils from allergic asthmatics and not non-allergic asthmatics responded to HRF. Of those allergic subjects who responded in vitro, methylcho- line sensitivity and symptoms of asthma were highly correlated [21]. Sampson et al have shown that production of HRF also is associated with clinical status of food allergy and atopic dermatitis [22]. Based on these observations, we believe that HRF may be an important element of the pathogenesis of asthmatic, allergic diseases. Since HRF is present in late phase reaction fluids in vivo, it may be contributing to mediator release that is found in the late response. Further understanding of the biology of HRF may help explain the varying severities of allergic disease. Although HRF has been extensively investigated for many years, most studies have been carried out in primary human or cultured cells. Currently, there is no established animal model available to further explore the function of HRF. One group from Taiwan generated HRF knockout mice by targeted gene disruption [23]. However, HRF knock out mice were embryonic lethal. Since HRF is ubiquitous and highly conserved, our approach has been to create an inducible HRF mouse model using the Tet-On system. Since we wanted to target HRF to the lungs, we used the CC10 promoter that is expressed in Clara cells of the lung epithelium. Here, we report the phenotype of this HRF-inducible mouse. Materials and Methods Ethics Statement All procedures performed on mice were in accordance with the National Institutes of Health guidelines for humane treatment of animals and were approved by the Johns Hopkins University Institutional Animal Care and Use Committee (ACUC). The ACUC protocol number is MO07M197. Transgenic TRE-HRF-EGFP plasmid construction The HRF transgenic plasmid was generated by the combination of three main components. The first component is the pTRE-tight vector, which contains a modified TRE (tet response element) controlling the inducible expression of the gene of interest. The second component is human HRF cDNA, which was cloned from U937 cells by RT-PCR and confirmed by sequencing. The third component is the pIRES2-EGFP vector (Clontech, Mountain View, CA). The IRES2 (internal ribosome entry site) allows the enhanced green fluorescent protein (EGFP) gene to be expressed individually as a reporter protein along with HRF in order to facilitate the recognition of expression of transgenic human HRF. Generation of transgenic mice Transgenic HRF mice were generated by pronuclear injection of the transgenic plasmid described above by the HRF Transgenic Mice Phenotype John Hopkins Transgenic Core. HRF transgenic mice were crossbred with CC10-rtTA mice on a C57BL/6 background to obtain double transgenic CC10-rtTA/HRF mice (transgene++) for the functional experiments. All experiments shown in these studies were completed during crossbreeding to reach pure background at generation 10. Therefore each protocol was completed with littermate control mice to account for generational variety. Mice were used at 6–11 weeks of age. All mice were housed in cages with microfilters in a specific pathogen-free environment. Identification of transgenic mice The presence or absence of the transgene in the resulting animals and their progeny was determined using tail DNA and PCR analysis. The primer sets used for PCR were EGFP-F, 59- GAC GTA AAC GGC CAC AAG TT-39; EGFP-R, 59-GAA CTC CAG CAG GAC CAT GT-39; TRE-HRF-F, 59-GTG TAC GGT GGG AGG CCT AT-39; TRE-HRF-R, 59-GTT TCC TGC AGG TGA TGG TT-39; and CC10-F, 59-ACT GCC CAT TGC CCA AAC AC-39; CC10-R, 59-AAA ATC TTG CCA GCT TTC CCC-39. The following PCR protocol was used: 95uC for 5 min; 30 cycles of 95uC for 45 sec, 62uC (EGFP and HRF) or 60uC (CC10) for 45 sec, and 72uC for 45 sec; and a final extension at 72uC for 5 min. Experimental design (1) Induction of transgene overexpression-Protocol I All TRE-HRF-EGFP transgenic mice and CC10 control mice were maintained on normal water until they were 6 weeks old. Then doxycycline (Dox) water at 1 mg/ml in 4% sucrose kept in dark bottles to prevent light-induced degradation was administrated for 3–4 weeks. Regular drinking water was given to littermate controls for comparison for the duration of the experiment (n = 4211 per group). (2) Ovalbumin (OVA) or PBS sensitization followed by OVA challenge-Protocol II The sensitization and challenge protocol was completed as previously described [24]. Briefly, all mice were admin- istrated Dox (1 mg/ml in 4% sucrose) in water on the first day of the experiment (day -7) and through out the challenge protocol to control for nonspecific Dox effects. Mice were divided into four groups (n = 629 per group): transgene++ and CC10 littermate sensitized with either OVA (Sigma, St Louis, MO) or PBS on day 0 by intraperitoneal (i.p.) injection of OVA (20 mg OVA adsorbed to 4 mg aluminum hydroxide) or PBS as a control and then boosted with OVA or PBS at day 5. Seven days later, all mice received a daily intra nasal challenge of OVA (20 mg) for three consecutive days. (3) OVA sensitization followed by OVA (or PBS) challenge- Protocol III To further explore the role of HRF in the allergic diathesis we design additional experiments that compared effects of HRF on mice that are sensitized with OVA and then challenged with OVA (as previously described in 2) or with sham challenge of PBS (n = 628 per group). The protocol was otherwise the same as previously described. Assessment of airway hyperresponsiveness (AHR) Airway hyperresponsiveness (AHR) was assessed by methylcho- line-induced airflow obstruction from conscious unrestrained mice placed in a whole body plethysmograph (model PLY 3211, Buxco Electronics Inc., Troy, New York, USA) [25]. In brief, mice were PLoS ONE | www.plosone.org 2 June 2010 | Volume 5 | Issue 6 | e11077