Analyses of Gnai3-iresGFP reporter mice reveal unknown Gαi3 expression sites

Abstract

Abstract Inhibitory G proteins (Gi proteins) are highly homologous but play distinct biological roles. However, their isoform-specific detection remains challenging. To facilitate the analysis of Gαi3 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 Gαi3. The expression of GFP was confirmed in spleen and thymus tissue by immunoblot analysis. Importantly, the GFP knock-in (ki) did not alter Gαi3 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 Gαi3 expression. In summary, the Gnai3- iresGFP reporter mouse represents an ideal tool for precise analyses of Gαi3 expression patterns and sites.