the minimal Tet-ON operator (Tetracyclin Responsive Element (TRE)-containing promotor). RNF5 transgenic (Tg) lines were then established by pronuclear injection and implantation in C57/BL6 recipient mice (see Methods for details). The rtTA RNF5 double transgenic animals (DTg) and their corresponding control littermates (RNF5 STg) were generated by crossing RNF5 STg with rtTA animals, expressing the tetracyclin responsive Transcriptional Activator under the control of the ubiquitous CMV-b-actin promotor (Fig. 2A; [25]. Overexpression of RNF5 protein was confirmed in different organs of the rtTA RNF5 DTg animals provided with doxycyclin in drinking water (Fig. 2B). Expression of RNF5 protein was evident in double transgenic animals, but not in doxycyclin-treated single transgenic animals (RNF5 STg) or untreated DTg animals. The RNF5 transgene is expressed at different levels depending on the organs analyzed. The higher level of RNF5 expression was seen in skeletal muscle where RNF5 transgene could be detected by straight western blot. RNF5 was expressed to a lesser extent in heart and in kidney where immunoprecipitation was required to detect the protein (Fig. 2B). Conversely, RNF5 levels were very low in liver and undetectable in brain, lungs and spleen (data not shown). This expression pattern is consistent with the low transcriptional levels of the rtTA activator and the differential RNF5 in ER-Related Disorders expression pattern described for the rtTA transgene [25]. These data suggest that RNF5 expression is tightly controlled in DTg mice and that the system is not subjected to transcriptional leakage in the absence of doxycyclin induction. Furthermore, the expression of RNF5 in skeletal muscles makes it a suitable system for studying its function in this organ, without restricting its expression to mature muscle fibers. Induction of RNF5 transgene leads to rapid weight loss and early onset of muscle wasting and kyphosis rtTA RNF5 DTg but not control animals subjected to doxycyclin treatment exhibited a significant weight loss as early as 2 wk post- induction (Figs. 2C, 2D). By 4 wk, clear phenotypic differences were evident between the double transgenic animals and their control littermates, including a significant decrease in body mass and visible kyphosis (Fig. 2C, 2D). After 5–6 wk of RNF5 overexpression, the phenotype was even more severe: the animals showed decreased activity and pelvic limb weakness. Death eventually occurred 7 weeks following initiation of doxycyclin treatment. Skeletal muscles were collected from double transgenic mice and analyzed for histopathologic changes. Compared with controls, double transgenic animals exhibit clear pathological Figure 2. Conditional expression of RNF5 in a transgenic mouse system. A Schematics depicting transgenic constructs used to overexpress RNF5 in mice. The rtTA transcriptional activator is expressed under the control of a CMV enhancer/chicken b-actin promoter. The RNF5 transgene is expressed under the control of a tetracycline-responsive promoter and is activated only in the presence of both the rtTA activator and doxycyclin. B. Tissue expression of RNF5 transgene in double transgenic animals. rtTA RNF5 DTg animals were treated with 2 mg/ml of doxycyclin in drinking water for 10 days and RNF5 protein levels were monitored in different organs. RNF5 expression was assessed using RNF5 polyclonal antibody, either by western blotting (skeletal muscle) or after immunoprecipitation (heart, kidney). a-tubulin was used as a loading control. In the case of heart and kidney, equal volumes of supernatants obtained following the immunoprecipitation were loaded and probed with a-tubulin. C. Comparison of a representative DTg animal overexpressing RNF5 after 4 wk of doxycyclin treatment with its control littermate. D. Weight curve comparison of RNF5- overexpressing DTg animals and their matching controls during doxycyclin treatment. The body mass of individual animals was monitored on a weekly basis for 6 wk. Graphs represent the change in body mass relative to the original weight of a single animal (n = 5). Note: In the same gender and age class, both experimental and control animals had the same external appearance and similar weight at the beginning of treatment. doi:10.1371/journal.pone.0001609.g002 PLoS ONE | www.plosone.org 4 February 2008 | Volume 3 | Issue 2 | e1609