Development of Transgenic Mouse Model Resources for Red Fluorescence-Based Genetically Encoded Indicator Studies - Scientific Abstract By expressing fluorescent reporters, such as green fluorescent protein (GFP) or red fluorescent protein (RFP) downstream of specific promoters or fusing them with endogenous genes, a range of cellular processes can be easily tracked and studied using fluorescence microscopy. Fluorescent reporters become even more powerful when they are tagged with a protein domain that transforms their function into an indicator of specific cellular processes, and they are typically referred to as ‘genetically encoded fluorescent indicators.’ Such indicators have been specifically designed by researchers to detect changes in ion concentration, membrane potential, and pH and have been used to generate transgenic mouse models for in vivo studies. The vast majority of these transgenic mice expressing genetically encoded fluorescent indicators utilize GFP as the fluorescent moiety. Unfortunately, major limitations of GFP-based indicators are phototoxicity and tissue autofluorescence which make it difficult to differentiate signal from the noise. Although RFP-based indicators can overcome these issues and they provide greater tissue penetration depth, there are not many animal-model resources currently available in the red emission range. The goal of this proposal is to develop mouse models expressing RFP-based indicators, characterize them, and make them available to the scientific community. We will utilize a novel clustered regularly interspaced short palindromic repeats (CRISPR)-based genome engineering approach called RE-CREATING (RE-engineering, with CRISPR, of previously Engineered Alleles To Insert New Genes), to generate two conditional knock-in mouse models. A popular and well-studied mouse strain that expresses a green genetically encoded fluorescent indicator will be used as the parental strain. The transgene locus in this parental strain will be re-engineered by swapping the GFP-based indicator for a RFP-based indicator, without disrupting any of the regulatory elements. The newly developed RFP-based indicator mouse models will be suitable to study any cell type of interest simply by crossing them with a Cre driver mouse of choice. Our models will enable imaging of dynamic intracellular processes such as changes in calcium activity and membrane potential, which can be challenging and/or inconsistent across tissues in currently available GFP-based models (due to phototoxicity and autofluorescence). We will use vascular physiology techniques (e.g., pressure myography and sharp microelectrode measurements of membrane potential) to characterize and validate the RFP-based indicator mouse models. Overall, this project will provide a much-needed resource to the scientific community and opens the door for imaging of dual indicator mice and/or allows for imaging of processes not traditionally possible with GFP-based mouse models.
