Abstract
Background: Vitamin A is essential for a wide range of life processes throughout embryogenesis to adult life. With the aim of developing an in vivo model to monitor retinoic acid receptor (RAR) transactivation real-time in intact animals, we generated transgenic mice carrying a luciferase (luc) reporter gene under the control of retinoic acid response elements (RAREs) consisting of three copies of a direct repeat with five spacing nucleotides (DR5). Methods: Transgenic mice carrying a RARE dependent luciferase reporter flanked with insulator sequence were generated by pronuclear injection. RARE dependent luciferase activity was detected by in vivo imaging or in tissue extracts following manipulations with RAR/retinoid X receptor (RXR) agonists, RAR antagonists or in vitamin A deficient mice. Results: We found a strong induction of luciferase activity in a time and dose dependent manner by retinoic acid as well as RAR agonists, but not by the RXR agonist (using n=4-6 per group; 94 mice). In addition, luciferase activity was strongly reduced in vitamin A-deficient mice (n=6-9; 30 mice). These observations confirm that luciferase activity was controlled by RAR activation in the RARE-luc mouse. Luciferase activity was detectable in various organs, with high activity especially in brain and testis, indicating strong retinoid signalling in these tissues. Conclusion: The RARE-luc transgenic mice, which enabled real-time in vivo assessment of RAR activation, will be useful in understanding the normal physiology of vitamin A, the role of retinoid signalling in pathologies as well as to evaluate pharmacological ligands for RARs.
Highlights
Vitamin A is required throughout embryogenesis to adult life, and proper levels of vitamin A derivatives, called retinoids, are crucial for normal morphogenesis during embryonic development [1,2,3,4]
We tested the ability of 1 μM all-trans-retinoic acid (ATRA) to induce transcription of a variety of different retinoic acid response elements (RAREs)-luciferase constructs in P19 embryonic carcinoma cells (DR5-RARE derived from either retinoic acid receptor (RAR)-β2 promoter or the CYP26A1 promoter, one or multiple copies of the response elements, different directions, etc.)
The construct used for generating transgenic mice consists of three RARE repeats, DR5 binding site derived from RAR-β2 promoter [41], coupled to a TATA-box from the herpes simplex virus thymidine kinase (TK) promoter and the luciferase reporter gene, flanked with an insulator sequence [42]
Summary
Vitamin A is required throughout embryogenesis to adult life, and proper levels of vitamin A derivatives, called retinoids, are crucial for normal morphogenesis during embryonic development [1,2,3,4]. The majority of the effects of retinoids are mediated by the binding of all-trans-retinoic acid (ATRA) as the endogenous retinoic acid receptors (RARs) ligand and 9-cis-13,14-dihydroretinoic acid (9CDHRA), as the endogenous ligand of the RXRs [7, 8]. With the aim of developing an in vivo model to monitor retinoic acid receptor (RAR) transactivation real-time in intact animals, we generated transgenic mice carrying a luciferase (luc) reporter gene under the control of retinoic acid response elements (RAREs) consisting of three copies of a direct repeat with five spacing nucleotides (DR5). RARE dependent luciferase activity was detected by in vivo imaging or in tissue extracts following manipulations with RAR/retinoid X receptor (RXR) agonists, RAR antagonists or in vitamin A deficient mice. Conclusion: The RARE-luc transgenic mice, which enabled real-time in vivo assessment of RAR activation, will be useful in understanding the normal physiology of vitamin A, the role of retinoid signalling in pathologies as well as to evaluate pharmacological ligands for RARs
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