Generation of transgenic mice to investigate the expression patterns, protein interactions and physiological functions of P2X4, P2X6 and P2X7 receptors
Zusammenfassung der Projektergebnisse
P2X receptors represent important targets for the development of novel drugs. However, investigation of their precise localization, molecular, and physiological functions has been challenging and therefore, they remain poorly understood. The aim of this project was to generate bacterial artificial chromosome (BAC) transgenic mouse models that enable more specific investigation of these receptors. In the originally planned conditional approach, these transgenic mice should enable visualization of the expression pattern of P2X4, P2X6, and P2X7 subunits by expression of a soluble EGFP reporter and, upon crossing with different Cre-expressing mice, tissue-specific over-expression of the respective tagged P2X subunit for functional and biochemical studies. The project was substantially held up for several reasons. Therefore, the original strategy was finally changed and a conventional approach was used to overexpress EGFP-tagged P2X7 receptors under the control of their endogenous promoter. Several mouse lines expressing two polymorphic variants of an EGFP-tagged P2X7 receptor were successfully generated. The EGFP-tagged receptors are efficiently expressed and can be directly visualized by fluorescence, confocal, and two-photon microscopy. The obtained lines show different expression levels but identical expression patterns with predominant expression in molecular layers of the cerebellum and dentate gyrus. Co-staining with different cellular markers revealed the presence of P2X7-EGFP in microglia, oligodendrocytes, and subpopulations of astrocytes in the brain and in the spinal cord. P2X7-EGFP is targeted to specific subcellular sites and, in combination with deglycosylation analysis, these results indicate an efficient transport of the EGFP-tagged P2X7 receptors to the plasma membrane. In pull-down experiments, the tagged receptors could be specifically purified and were shown to assembly with endogenous P2X7 subunits. Mice from the strain with the highest transgene expression (about 10 fold over-expression) develop significant motor deficits already at a very young age suggesting a pathophysiological effect of P2X7R over-expression in motor coordination. In conclusion, we generated a novel mouse model that should be able to answer many of the unresolved questions in the P2X7 receptor field. The high interest in this mouse model is documented by a poster price and a travel grant (together with an invited talk) received at the "Purines 2012" conference in Fukuoka, Japan. Detailed analysis of these mice in collaboration with members of the Research Unit as well as external partners is currently ongoing.
