Abstract
Retinoic acid (RA) is an intriguing metabolite that is necessary for embryonic development and differentiation in vertebrates. The present protocol demonstrates how to image RA activities indirectly in mammalian cells with ligand-activatable single-chain bioluminescence (BL) probes. We introduce 13 different molecular designs for characterizing an efficient single-chain probe that quantitatively visualizes RA activities with significant sensitivity. The key components included in the probes are (i) the N- and C-terminal fragments of artificial luciferase 16 (ALuc16), (ii) the ligand-binding domain of human retinoic acid receptor α (RAR LBD), and (iii) an LXXLL motif derived from common coactivators of nuclear receptors. The probe is highly selective and sensitive to all-trans-RA (at-RA) in animal cells. This protocol exemplifies quantitative imaging of the RA levels in serum and cerebrospinal fluid with a linear range in two orders. The present protocol is an important addition to conventional techniques on quantitative imaging of endogenous at-RA levels in live mammalian cells.
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Acknowledgements
This work was partly supported by JSPS KAKENHI Grants: Numbers 15KK0029, 17H01215, 20 K21851, and 21H04948.
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© 2022 The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature
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Kim, SB., Fujii, R., Paulmurugan, R. (2022). Quantitative Imaging of Retinoic Acid Activities in Living Mammalian Cells. In: Kim, SB. (eds) Bioluminescence. Methods in Molecular Biology, vol 2525. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-2473-9_8
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DOI: https://doi.org/10.1007/978-1-0716-2473-9_8
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