Generation of T cell specific Annexin1 transgenic mice: a novel tool to study rheumatoid arthritis.

Nick Paschalidis, Robert Hannon, Mauro Perretti, Fulvio D’Acquisto
William Harvey Research Institute, London, United Kingdom

We have recently shown that exogenous human recombinant Annexin-1 (Anx-A1) modulates T cell activation by increasing the strength of T Cell Receptor (TCR) signalling. The aim of this study is to determine the effects of increased levels of Anx-A1 in T cells in vivo by generating transgenic mice overexpressing this protein selectively in T cells.

To this aim we used the VACD2 vector that contains the Locus Control Region (LCR) of the human CD2 gene allowing tissue specific, copy-number dependent and integration site independent expression of the linked gene and the proximal CD2 promoter that allows the expression in 95% of thymocytes and peripheral T cells. Murine Anx-A1 cDNA was amplified from RAW 246.5 cells by standard PCR. In order to differentiate the endogenous Anx-A1 from the transgenic one, we designed primers that incorporated the FLAG epitope at the carboxyl-terminal of the translated protein. The FLAG-tagged Anx-A1 was cloned into the SmaI cloning site of the VACD2 vector and the resultant construct injected in the oocytes of CBAxC57BL/6 pseudo pregnant female after removal of the vector prokaryotic sequences (Transgenic core facility, ICMS, QMUL, London). We have obtained 22 mice and identified four transgenic founders from which two showed mendelian transmission of the transgene in the F1 generation.

We are currently backcrossing the transgenic founders and examining the phenotype of these mice. Once we establish the colony we will examine possible macroscopic differences compared to littermate control mice such as size and cellularity of lymphoid organs and number of circulating T cells. Finally we will also investigate possible changes in early and late TCR signalling and analyze the in vivo relevance of these findings using mouse models of autoimmune diseases such as rheumatoid arthritis or multiple sclerosis.