Dataset Information

Introducing a new reporter gene, membrane-anchored Cypridina luciferase, for multiplex bioluminescence imaging.

ABSTRACT: Bioluminescence reporter gene imaging is a robust, high-throughput imaging modality that is useful for tracking cells and monitoring biological processes, both in cell culture and in small animals. We introduced and characterized a novel bioluminescence reporter-membrane-anchored Cypridina luciferase (maCLuc)-paired with a unique vargulin substrate. This luciferase-substrate pair has no cross-reactivity with established d-luciferin- or coelenterazine-based luciferase reporters. We compare maCLuc with several established luciferase-based reporter systems (firefly, click beetle, Renilla, and Gaussia luciferases), using both in vitro and in vivo models. We demonstrate the different imaging characteristics of these reporter systems, which allow for multiplexed-luciferase imaging of 3 and 4 separate targets concurrently in the same animal within 24 h. The imaging paradigms described here can be directly applied for simultaneous in vivo monitoring of multiple cell populations, the activity of selected signal transduction pathways, or a combination of both constitutive and inducible reporter imaging.

SUBMITTER: Moroz MA

PROVIDER: S-EPMC8020342 | biostudies-literature | 2021 Jun

REPOSITORIES: biostudies-literature

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Publications

Introducing a new reporter gene, membrane-anchored <i>Cypridina</i> luciferase, for multiplex bioluminescence imaging.

Moroz Maxim A MA Zurita Juan J Moroz Anna A Nikolov Ekaterina E Likar Yury Y Dobrenkov Konstantin K Lee Jason J Shenker Larissa L Blasberg Ronald R Serganova Inna I Ponomarev Vladimir V

Molecular therapy oncolytics 20210317

Bioluminescence reporter gene imaging is a robust, high-throughput imaging modality that is useful for tracking cells and monitoring biological processes, both in cell culture and in small animals. We introduced and characterized a novel bioluminescence reporter-membrane-anchored <i>Cypridina</i> luciferase (maCLuc)-paired with a unique vargulin substrate. This luciferase-substrate pair has no cross-reactivity with established d-luciferin- or coelenterazine-based luciferase reporters. We compare ...[more]

PMID: 33851009

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Project description:PurposeFirefly 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.MethodsBenzene ring (14)C(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.ResultsRadiolabeled 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 (14)C-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 (14)C-D-luciferin in firefly luciferase-expressing tissues could be observed in vivo.ConclusionsThe data obtained with (14)C-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.

Dataset Information

Introducing a new reporter gene, membrane-anchored Cypridina luciferase, for multiplex bioluminescence imaging.

Publications

Introducing a new reporter gene, membrane-anchored <i>Cypridina</i> luciferase, for multiplex bioluminescence imaging.

Similar Datasets

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