Environmental Management Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba, Japan.
Molecular Imaging Program at Stanford, Stanford University School of Medicine, Palo Alto, CA, USA.
Methods Mol Biol. 2022;2525:111-122. doi: 10.1007/978-1-0716-2473-9_8.
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.
视黄酸（RA）是一种有趣的代谢物，对于脊椎动物的胚胎发育和分化是必需的。本方案演示了如何使用配体激活的单链生物发光（BL）探针间接对哺乳动物细胞中的 RA 活性进行成像。我们介绍了 13 种不同的分子设计，用于表征高效的单链探针，该探针具有显著的灵敏度，可定量可视化 RA 活性。探针中包含的关键组件有（i）人工荧光素酶 16（ALuc16）的 N 和 C 末端片段，（ii）人视黄酸受体α（RAR LBD）的配体结合域，以及（iii）核受体共激活因子衍生的 LXXLL 基序。该探针在动物细胞中对全反式视黄酸（at-RA）具有高度选择性和敏感性。该方案示例说明了通过线性范围在两个数量级内对血清和脑脊液中的 RA 水平进行定量成像。与传统的活哺乳动物细胞内内源性 at-RA 水平的定量成像技术相比，本方案是一个重要的补充。
