Functional validation of P2RX7 as a susceptibility marker for depression using humanized mouse mutants

Recent linkage and association studies suggest P2RX7 as a novel susceptibility gene for major depressive disorder (MDD) and bipolar disorder (BP). A non-synonymous SNP has been shown to be associated with reduced calcium influx and is likely to affect P2RX7 oligomerization and interaction. To study the functional relevance of the polymorphism in an appropriate in vivo model, we generated humanized mouse mutants in which the murine P2RX7 gene was substituted by the wild-type or the disease-associated isoform of human P2RX7. A knock-in approach based on homologous recombination in embryonic stem (ES) cells was used. ES cell clones were screened by Southern blot analysis. Positive clones were injected into blastocysts and chimeric animals were screened for germline transmission by PCR. In case of the mutant variant of human P2RX7 we obtained two correctly recombined ES cell clones and 24 chimeras in total. In terms of the wild-type variant we identified four positive clones which resulted in a total of 23 chimeras. In both cases the humanized allele was transmitted through the germline. The correct expression of the human P2RX7 variants in the brain of mutant mice was confirmed by ISH using a human-specific probe. Using these mouse lines, we will be able to functionally validate the human association data. Future analyses will involve molecular, endocrinological, electrophysiological and behavioural paradigms.