HRF Transgenic Mice Phenotype Figure 2. Transgenic Protein and mRNA Expression for Mice in Protocol I. In all cases CC10 are control mice either fed Dox (Dox) or fed regular water (no Dox). Transgenic mice (++) are either fed Dox (Dox) or are on regular water (no Dox). Each group has between 4 and 11 mice (backcrosses 5–8). Panel A depicts the relative amount of HRF protein based on densitometric analysis of Western blots. Panel B is the relative amount of GFP protein based on Western blots. Relative protein levels are expressed as a function of the positive control of each protein. Panel C is a representative Western blot in the transgenic mice (++) or littermate controls (CC10) in the presence of doxycycline (Dox) or absence of doxycycline (ND). Panel D shows mRNA for GFP relative to the housekeeping gene, beta actin. doi:10.1371/journal.pone.0011077.g002 capacity of each cell. The images in Figure 5 indicate that either could be true. HRF is an abundant cellular protein and has been implicated in cell cycle-related activities [30], but there are numerous reports of its extracellular function [2,8–10,15–18]. In order to demonstrate that HRF was additionally secreted in our mouse system, we concentrated BAL samples 50-fold from OVA challenged transgenic mice and compared to concentrated BAL from OVA challenged CC10 littermate controls. The results of the Western blot are shown in Figure 6. HRF expression levels in the transgenic mice is greater than that from CC10 littermate controls. While HRF is not visible in the controls due to the level of detection, these expression levels of HRF in all BAL from transgenic mice are consistent with the literature. In that case, a 100-fold concentra- tion was necessary to detect HRF in human BAL of asthmatic patients [18]. This demonstrates that HRF is secreted and available to act extracellular to activate cells. Thus, our transgenic mouse provides both an intracellular and extracellular expression of HRF. Lung Histology. OVA sensitized and challenged mice demonstrated a marked cellular infiltrate in the bronchus when stained with hematoxilin and eosin, as shown in Figure 7. Eosinophils and enlarged and activated macrophages were seen in transgenic++ mice sensitized and challenged with OVA (++/ OVA). This pattern was observed in comparison to CC10 littermate controls treated the same way (CC10/OVA), as well PLoS ONE | www.plosone.org 5 June 2010 | Volume 5 | Issue 6 | e11077