The quantitative data of continuous variables were expressed as mean 6 S.E.M of each group. The statistical difference between experimental groups was determined by Student’s t test. A p value of less than 0.05 was considered statistically significant. Results Over Expression of Transgene and Phenotypic Analysis (Protocol I) Transgene expression. The schematic of the physical map of the transgene is shown in Figure 1. We specifically did not analyze copy numbers and integration sites of the transgene. Using a combination of endonuclease digestion, PCR and nucleotide sequencing, transgene integration sites in the genome can be determined. However, knowing where the transgene is does not prevent its segregation from its surrounding genes in the offspring, and there are no practical values in the function of the transgene. Ultimately the expression and function of the transgene needs to be tested experimentally. The same is true with the copy numbers of the transgene. Additionally, correlation between copy numbers and expression and function of a transgene is not very clear. After crossbreeding, both transgene++ and CC10 control mice were treated with Dox at 1 mg/ml for 3–4 weeks. Regular drinking water was also given to littermate controls for comparison. Transgene expression was verified by both Western blot and mRNA analysis of lung tissue. First, lung tissue lysates were analyzed by Western blot for both EGFP and HRF expression, as shown in Figure 2. The homology between mouse and human HRF yields a cross-reaction and therefore Western blots could not distinguish between native and transgenic HRF expression, therefore, GFP was used as an additional marker of transgene expression. Expression of HRF (Figure 2, Panel A) as well as over expression of GFP (Figure 2, Panel B) in Dox treated mice indicated that the Tet on system was operating effectively. However we did see a modest, but not statistically significant nonspecific Dox effect on HRF expression in CC10 mice. Furthermore, there was a Dox-independent induction of HRF when CC10 and ++ mice were fed with normal water (p = 0.02). However, GFP expression (Panels B and D) was not significantly increased among these groups. Therefore, we do not think there is evidence of promoter leakiness. Nevertheless, subsequent studies were done comparing transgene++ and CC10 littermate controls all fed Dox. RT-PCR analysis of lung tissues for EGFP expression confirmed these results (Figure 2, Panel D). Of note, a representative Western blot in the transgene++ or CC10 littermate controls in the presence or absence of doxycycline is shown in Figure 2, Panel C. Phenotypic analysis. BAL cells were examined and there was a significant increase in total cell counts in transgene++ mice compared to CC10 mice following dox induction and the change was primarily due to an increase in macrophages (p = 0.04, Figure 3). Of note, there are significant differences (p,0.001) between transgenic ++ mice and CC10 littermate controls not fed Dox in both total cells and macrophages. This may be due to the increased HRF expression noted in Figure 2. However, given that Figure 1. Schematic of the Physical Map of the HRF Transgene. The plasmid construction is described in the Materials and Methods. TRE (tet response element), pIRES (internal ribosome entry site), EGFP (enhanced green fluorescent protein). doi:10.1371/journal.pone.0011077.g001 HRF Transgenic Mice Phenotype the Dox induction between transgenic ++ and control CC10 mice was significantly different, we proceeded with experiments in which all mice were fed Dox, as previously noted. Due to the increase in macrophages, we investigated mRNA levels for MCP- 1. MCP-1 is known to be involved in the initiation of inflammation [28]. As shown in the insert of Figure 3, there was a significant increase in mRNA levels for MCP-1 in transgenic++ mice fed Dox as compared to CC10 mice fed dox (p = 0.04) and to transgenic++ mice fed regular water (p = 0.05). Markers of TH2 inflammation were also examined, including serum and BAL IgE levels, eotaxin, IL4, IL5 and IL13 expression in lung tissues, but there were no differences seen (data not shown). This is not surprising based on the lack of TH2 cellular infiltrate in the lungs of the transgene++ mice (Figure 3), and the previously published studies. Specifically, Teshima et al found that HRF caused eosinophil recruitment in sensitized but not normal mice [29]. Additionally, Kang et al found that in vivo administration of HRF increased total and Ag-specific Ig synthesis, but these studies were done in the more conventionally allergic BALB/c mice [17]. Thus, we had a modest phenotype when the HRF transgene was turned on, but it was not the allergic phenotype we initially expected. Therefore, we proceeded to use our transgene++ mice in OVA sensitization and challenge experiments to see if HRF enhanced the allergic phenotype. OVA (or PBS) Sensitization followed by OVA Challenge (Protocol II) The sensitization and challenge protocol was essentially the same as described by Brusselle et al [24] and is outlined in Material and Methods section. All mice were fed Dox 7 days prior to and throughout the challenge protocol to control for nonspecific Dox effects. The mice were divided into 4 groups: transgene++ and CC10 littermates sensitized with either OVA or PBS as a control (n = 629 per group). This model has been used extensively and has generated brisk and consistent TH2 responses in the murine lung [26]. After intranasal challenge, the phenotype of each mouse was also assessed by BAL cell counts, histopathology of bronchi and trachea, serum and BAL IgE levels, allergic cytokine gene and protein expression in the BAL and/or lung tissue. Transgene expression. Transgene expression was confirmed in each mouse as previously described and is shown in Figure 4. Of note, HRF protein levels (Panel A) were increased following OVA sensitization and challenge compared to PBS sensitized OVA challenged litter mate controls in transgene++ mice (p = 0.006) and in CC10 controls (p = 0.02). The increases in HRF are endogenous, and not promoter driven transgenic HRF based on the similar GFP levels in the groups (Figure 4, Panel B). This indicates that OVA sensitization increases naturally occurring HRF, and supports HRF’s role in the antigen driven allergic phenotype. Lung cross-sections were subjected to immunoflourescent staining to visualize HRF expression localized to the lung epithelium. Immunoflourescent staining shown in Figure 5, illustrates that HRF is up-regulated in the epithelium of transgene++ mice fed Dox (Panels C and D) compared to littermate controls (Panel A). Furthermore, OVA sensitization up- regulates HRF in the lung epithelium (Panel D, compared to Panel C). Nonspecific staining with no primary antibody (Panel B), as well as additional experiments with an irrelevant antibody (not shown) demonstrated no non-specific staining. The increase in HRF expression could be due to an increase in the number of Clara cells following antigen challenge or an increase in the PLoS ONE | www.plosone.org 4 June 2010 | Volume 5 | Issue 6 | e11077