The CRE-Luc lines can serve as a source of primary cells with the GPCR reporter in its native environment. Therefore in vitro studies can be first performed followed by in vivo studies.
In vitro Studies (Primary Cells and ex vivo assays)
Initial analysis of ligand activation can first be confirmed in CRE-Luc primary cell cultures. For example, CRE-Luc cell cultures support GPCR receptor specificity assays, e.g., through the use of RNAi or ligand competition assays; these are an important validation step since potentially any receptor (or combination of receptors) can be activated by a single ligand.
After profiling ligand activation in primary cells, more complex tissue profiles can assayed either ex vivo or through isolated tissue homogenates that assay luciferase enzyme levels. Tissue homogenate analysis is time consuming, but when used with compound dosing in whole animals, can generate a detailed, tissue specific, and quantitative ligand activation profile.
In vivo Studies (Whole Animal)
After activation profiles are established from primary cells and tissues, then ligand profiles can be assayed in whole animals by bioimaging and incorporating dose response and time course assays. Data analysis can occur in the same day as the imaging session which allows unknown endpoints or results in the assay to be defined as the study progresses. This feature impacts flexibility in the animal study and can save significant time in avoiding repetitive studies to capture overlooked data. The whole animal bioimaging assay format can quantitatively define the site and magnitude of ligand activation plus support a quantitative comparison of similar compounds for selection of optimal lead structures and SAR.