Protein kinase A (PKA) is a key signaling molecule that has diverse functions, from regulating metabolism and brain activity to guiding development and cancer progression. FLIM-compatible A Kinase Activity Reporter mice (FL-AK, FLIM-AKARflox) express the FLIM-AKAR optical fluorescent reporter in a Cre-dependent manner. Cre recombinase-mediated excision of a loxP-flanked Stop cassette enables the observation of protein kinase A (PKA) activity with cellular resolution in real time. The design of FLIM-AKARflox mice is based on the FLIM-compatible PKA activity reporter FLIM-AKAR developed by Chen et al., 2016 (PMID: <a href="https://pubmed.ncbi.nlm.nih.gov/24765076/" target="_blank">26941646</a>). Compared with viral delivery or in utero electroporation strategies, the knock-in FLIM-AKARflox mice avoid invasive experimental methodologies, make the expression consistent, and allow easier targeting of sparsely expressed cell types. The Fluorescence Lifetime Imaging Microscopy compatible A Kinase Activity Reporter (FLIM-AKAR) consists of a donor fluorophore of monomeric enhanced green fluorescent protein (meGFP) and an acceptor fluorophore of dark yellow fluorescent protein (sREACH). When PKA phosphorylates the substrate consensus region within the linker region, the resulting phosphopeptide binds to the FHA1 phosphopeptide binding domain that is also in the linker region, thus bringing the donor and acceptor fluorophores closer together. This results in increased FRET and decreased fluorescence lifetime. This conformational change can also be reversed by phosphatases, which release the phosphopeptide from its binding domain through dephosphorylation. As such, FLIM-AKAR is a phosphorylation substrate reporter that reports the balance between PKA and phosphatase. Successful reporting of PKA activity in response to neuromodulator G protein-coupled receptor (GPCR) activation in specific cell types in brain slices and in freely moving mice has been demonstrated. Crosses with Emx1IREScre mice (Stock No. <a href="https://www.jax.org/strain/005628" target="_blank">005628</a>) labels excitatory neurons (and a small subset of glia) in the neocortex and hippocampus of progeny.

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FLIM-compatible A Kinase Activity Reporter (FL-AK, FLIM-AKARflox) mice express the FLIM-AKAR optical fluorescent reporter in a Cre-dependent manner. Cre recombinase-mediated excision of a loxP-flanked Stop cassette enables the observation of protein kinase A (PKA) activity with cellular resolution in real time. This strain has been useful in revealing PKA activity dynamics in genetically defined cell types via fluorescence lifetime imaging microscopy (FLIM) or photometry in brain slices and in vivo.

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