| CUEDC2 (CUE domain containing 2) protein contains a CUE domain which is small, moderately conserved ubiquitin-binding domain of 40 amino acids. CUE domain is found in a variety of eukaryotic proteins. These proteins are involved in the recognition of monoubiquitin and polyubiquitin as well as in facilitating intramolecular monoubiquitination.The previous study of our laboratory showed that CUEDC2 interacts with progesterone receptor (PR) and promotes progesterone-induced PR degradation through ubiqutin-proteasome pathway (The EMBO Journal,2007). Also, CUEDC2 acts as an adaptor protein to target IKK for dephosphorylation and inactivation by recruiting GADD34/PP1, and represses activation of the transcription factor NF-?B by decreasing phosphorylation and activation of IKK (Nature Immunology, 2008). Recently, through a study of specimens from 449 subjects with breast cancer, we found that CUEDC2 is highly expressed in human breast cancer and suggested CUEDC2 is a crucial determinant of resistance to endocrine therapies in breast cancer (Nature Medicine, 2011). However, the function of CUEDC2 in tumorigenesis remains largely unknown.In this study, we established a conditional transgenic mice model with specific expression of CUEDC2 in mammary gland by Tet-on system. The Tet-On system has been widely used for controlling gene expression in vivo and in vitro by administration of derivatives of tetracycline (Tet), such as doxcycline (Dox). This system consists of two constructs, one expressing the tetracycline transactivator (rtTA) under a tissue-specific promoter, and the other expressing the interested gene under a tetracycline responsive element (TRE). In this system, the rtTA protein is capable of binding the operator only when bound by doxycycline. Thus the introduction of doxycyline to the system initiates the transcription of the genetic product. First, we constructed a CUEDC2 transgenic expression vector. After verification of CUEDC2 expression in MCF7-rtTA cells, the vector was digested by restriction enzymes, and the DNA fragments were microinjected into fertilized eggs of mice. Then the manipulated embryos were transferred into the oviducts of pseudopregnant recipient female mice, and the pups of 3 founders were identified by PCR analysis. Next, CUEDC2 transgenic mice were crossed with the MMTV-rtTA transgenic mice for CUEDC2/rtTA double transgenic mice. The CUEDC2 expression of the offsprings was analyzed by PCR, RT-PCR and Western blot assays. Our results showed that twelve mice were identified as carrying copies of CUEDC2 gene. Immunohistochemical analysis and Western blotting data showed that the transgene of CUEDC2 was specific expressed in mammary gland in 2 transgenic mouse lineages.In conclusion, we established a CUEDC2/rtTA double transgenic mouse model, which will facilitate the investigation of the biological function of CUEDC2 in vivo... |
