Uptake kinetics and biodistribution of 14C-d-luciferin—a radiolabeled substrate for the firefly luciferase catalyzed bioluminescence reaction: impact on bioluminescence based reporter gene imaging
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Firefly luciferase catalyzes the oxidative decarboxylation of d-luciferin to oxyluciferin in the presence of cofactors, producing bioluminescence. This reaction is used in optical bioluminescence-based molecular imaging approaches to detect the expression of the firefly luciferase reporter gene. Biokinetics and distribution of the substrate most likely have a significant impact on levels of light signal and therefore need to be investigated.
Benzene ring 14C(U)-labeled d-luciferin was utilized. Cell uptake and efflux assays, murine biodistribution, autoradiography and CCD-camera based optical bioluminescence imaging were carried out to examine the in vitro and in vivo characteristics of the tracer in cell culture and in living mice respectively.
Radiolabeled and unlabeled d-luciferin revealed comparable levels of light emission when incubated with equivalent amounts of the firefly luciferase enzyme. Cell uptake assays in pCMV-luciferase-transfected cells showed slow trapping of the tracer and relatively low uptake values (up to 22.9-fold higher in firefly luciferase gene-transfected vs. nontransfected cells, p = 0.0002). Biodistribution studies in living mice after tail-vein injection of 14C-d-luciferin demonstrated inhomogeneous tracer distribution with early predominant high radioactivity levels in kidneys (10.6% injected dose [ID]/g) and liver (11.9% ID/g), followed at later time points by the bladder (up to 81.3% ID/g) and small intestine (6.5% ID/g), reflecting the elimination routes of the tracer. Kinetics and uptake levels profoundly differed when using alternate injection routes (intravenous versus intraperitoneal). No clear trapping of 14C-d-luciferin in firefly luciferase-expressing tissues could be observed in vivo.
The data obtained with 14C-d-luciferin provide insights into the dynamics of d-luciferin cell uptake, intracellular accumulation, and efflux. Results of the biodistribution and autoradiographic studies should be useful for optimizing and adapting optical imaging protocols to specific experimental settings when utilizing the firefly luciferase and d-luciferin system.
KeywordsOptical imaging Luciferase d-Luciferin Reporter Genes Biodistribution
firefly luciferase gene
fetal bovine serum
adenovirus carrying fluc under the control of the cytomegalie virus promoter
- 293T Luc cells
293T cells expressing the Firefly luciferase gene
percent injected dose/gram
This work was supported by National Cancer Institute (NCI) Small Animal Imaging Resource Program (SAIRP) grant R24 CA93862, NCI ICMIC P50 CA114747 (SSG), and NIH R01 CA82214 (SSG). FB was supported by a grant of the German Research Foundation (DFG), a grant by the Bayerisch-Kalifornische Hochschulinitiative (BACATEC), and the Friedrich Baur Stiftung. We also thank Xenogen for purchasing 14C(U)-labeled d-luciferin from Moravek Biochemicals. The reported experiments comply with the current laws of the USA inclusive of ethics approval.
Conflict of interest statement
All authors state that they have no conflict of interests.
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