Analyses of Gnai3-iresGFP reporter mice reveal unknown Gα_i3 expression sites

Inhibitory G proteins (G_i proteins) are highly homologous but play distinct biological roles. However, their isoform-specific detection remains challenging. To facilitate the analysis of Gα_i3 expression, we generated a Gnai3- iresGFP reporter mouse line. An internal ribosomal entry site (IRES) was inserted behind the stop-codon of the Gnai3 gene to initiate simultaneous translation of the GFP cDNA together with Gα_i3. The expression of GFP was confirmed in spleen and thymus tissue by immunoblot analysis. Importantly, the GFP knock-in (ki) did not alter Gα_i3 expression levels in all organs tested including spleen and thymus compared to wild-type littermates. Flow cytometry of thymocytes, splenic and blood cell suspensions revealed significantly higher GFP fluorescence intensities in homozygous ki/ki animals compared to heterozygous mice (+/ki). Using cell-type specific surface markers GFP fluorescence was assigned to B cells, T cells, macrophages and granulocytes from both splenic and blood cells and additionally blood-derived platelets. Moreover, immunofluorescent staining of the inner ear from knock-in mice unraveled GFP expression in sensory and non-sensory cell types, with highest levels in Deiter's cells and in the first row of Hensen's cells in the organ of Corti, indicating a novel site for Gα_i3 expression. In summary, the Gnai3- iresGFP reporter mouse represents an ideal tool for precise analyses of Gα_i3 expression patterns and sites.