These previous studies have allowed for a better understanding of the biology of transplant rejection

e selection. -selection, one of the two major checkpoints during T cell development, ensures successful production of a TCR chain. Therefore, mice having defective gene rearrangement, such as Rag1/2 deficiency or scid mutation, could not pass beyond the checkpoint, resulting in a strict developmental arrest at the DN3 stage. However, the following treatments have been shown to allow checkpoint bypass without TCR protein synthesis: irradiation-induced PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19740122 DNA damage, inhibition of apoptosis, or forced activation of pre-TCR signaling. Indeed, constitutively active mutations in signaling molecules such as Lck, Ras and Rac could also induce DN-DP transition independent of TCR gene rearrangement. Besides a gene knockout approach, gain-of-function analysis, as typified by transgenic overexpression, is also useful to explore gene function. In this article, we have investigated the function of RhoH by utilizing RhoH transgenic mice under the control of the CD2 promoter. Overexpression of RhoH in thymocytes did not interfere with T cell development significantly, however, we found that excess RhoH resulted in bypass of the -selection checkpoint, allowing differentiation from DN to DP without TCR recombination in vivo. This DN to DP transition was also observed by in vitro culture, and was dependent on Lck. Since RhoH facilitates activation of Lck and Zap70, excess amounts of RhoH protein could initiate Lckdependent activation of pre-TCR signaling in the absence of pre-TCR complexes. Materials and Methods Ethics statements Animal experiments were approved by the Animal Care and Use Committee of the National Center for Global Health Medicine Research Institute and conducted in accordance with institutional procedures. All efforts were made to minimize suffering. Animals RhoHtg mice were generated by the microinjection of VA hCD2-HA-RhoH vector into the pronuclei of fertilized eggs using standard procedures. Previous studies have shown VA vector containing human CD2 promoter/T cell specific transcriptional enhancer directs the expression of transgenes in mice to the T cell lineage. The embryos were transferred to the oviducts of pseudopregnant ICR Roscovitine web female mice. Established mouse lines were maintained after 10 generations of backcrossing to C57BL/6J, and housed under specific pathogen-free conditions in accordance with institutional guidelines. Mice were sacrificed by cervical dislocation to dissect organs out. Reagents and antibodies Rat monoclonal and mouse monoclonal antibodies to HA were purchased from Roche and Recenttec, respectively. Anti Phospho-Src family and Phospho-ERK1/2 were purchased from Cell 2 / 13 RhoH Can Bypass the Pre-TCR Checkpoint signaling. RhoH mouse monoclonal antibody was purchased from Novus Biologicals. Monoclonal antibodies to CD2, CD4, CD5, CD25, CD44, PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19740489 CD62L, NK1.1, TCRb, TCRgd and Ter-119, and control antibodies for Armenian hamster IgG and rat IgG were purchased from eBioscience. These antibodies or reagents were directly coupled to allophycocyanin, APC/cyanine 7, Brilliant Violet 421, fluorescein isothiocyanate, phycoerythrin, PE/Cy7, or biotin. Recombinant murine interleukin -7 was purchased from R&D Systems. Lck inhibitor III was purchased from CALBIOCHEM. Flow cytometry To obtain single cell suspensions, lymphoid tissues from 4 to 8 week old mice were crushed and passed through 42 m pore nylon mesh. 1×106 cells were stained with saturating concentrations of indicated antibodies for 30 min at 4C, washed in 200 l of stai