To monitor cGMP signals in vivo, we have generated and characterized transgenic mouse lines expressing the fluorescence resonance energy transfer (FRET)-based cGi500 sensor (cGMP indicator with an EC50 value for cGMP of 500 nM, [1]). One mouse line produced by random transgenesis expresses cGi500 in vascular and visceral smooth muscle. Other mouse lines were created by targeted modification of the ROSA26 locus; these lines either show ubiquitous cGi500 expression, or they can be used for Cre/lox-dependent, tissue-specific cGi500 expression. These cGi500-transgenic mice are healthy and fertile, and do not show obvious adverse phenotypes.
Primary cells isolated from cGi500-expressing mice, including platelets, neural cells, and smooth muscle cells were used for FRET imaging experiments. NO-induced cGMP synthesis via soluble guanylyl cyclase was observed in the majority of cell types, while cGMP responses to atrial or C-type natriuretic peptides differed between cell types. Moreover, NO-induced cGMP elevations could be observed in the vasculature of retinas isolated from cGi500-expressing animals. Importantly, the feasibility of FRET-based cGMP imaging in living animals was demonstrated by intravital microscopy of anesthetized cGi500-transgenic mice. By epifluorescence FRET microscopy we could visualize transient NO-induced cGMP signals in arterial walls of the cremaster muscle. Furthermore, we used multi-photon microscopy in a dorsal skinfold chamber model and were able to record NO-elicited FRET changes in subcutaneous vessels.
