title: Fluorophore-NanoLuc BRET Reporters Enable Sensitive In Vivo Optical Imaging and Flow Cytometry for Monitoring Tumorigenesis.
abstract: Fluorescent proteins are widely used to study molecular and cellular events, yet this traditionally relies on delivery of excitation light, which can trigger autofluorescence, photoxicity, and photobleaching, impairing their use in vivo. Accordingly, chemiluminescent light sources such as those generated by luciferases have emerged, as they do not require excitation light. However, current luciferase reporters lack the brightness needed to visualize events in deep tissues. We report the creation of chimeric eGFP-NanoLuc (GpNLuc) and LSSmOrange-NanoLuc (OgNLuc) fusion reporter proteins coined LumiFluors, which combine the benefits of eGFP or LSSmOrange fluorescent proteins with the bright, glow-type bioluminescent light generated by an enhanced small luciferase subunit (NanoLuc) of the deep-sea shrimp Oplophorus gracilirostris. The intramolecular bioluminescence resonance energy transfer that occurs between NanoLuc and the fused fluorophore generates the brightest bioluminescent signal known to date, including improved intensity, sensitivity, and durable spectral properties, thereby dramatically reducing image acquisition times and permitting highly sensitive in vivo imaging. Notably, the self-illuminating and bifunctional nature of these LumiFluor reporters enables greatly improved spatiotemporal monitoring of very small numbers of tumor cells via in vivo optical imaging and also allows the isolation and analyses of single cells by flow cytometry. Thus, LumiFluor reporters are inexpensive, robust, noninvasive tools that allow for markedly improved in vivo optical imaging of tumorigenic processes.
authors.affiliation: Department of Tumor Biology, Moffitt Cancer Center and Research Institute, Tampa, Florida.
authors.forename: Franz X
authors.initials: FX
authors.lastname: Schaub
authors.affiliation: Department of Cancer Biology, The Scripps Research Institute, Scripps Florida, Jupiter, Florida.
authors.forename: Md Shamim
authors.initials: MS
authors.lastname: Reza
authors.affiliation: Department of Pharmacological & Physiological Science, School of Medicine, Saint Louis University, St. Louis, Missouri.
authors.forename: Colin A
authors.initials: CA
authors.lastname: Flaveny
authors.affiliation: Department of Tumor Biology, Moffitt Cancer Center and Research Institute, Tampa, Florida.
authors.forename: Weimin
authors.initials: W
authors.lastname: Li
authors.affiliation: UNC Biological and Biomedical Sciences Graduate Program, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina.
authors.forename: Adele M
authors.initials: AM
authors.lastname: Musicant
authors.affiliation: Scripps Graduate Program, The Scripps Research Institute, Scripps Florida, Jupiter, Florida.
authors.forename: Sany
authors.initials: S
authors.lastname: Hoxha
authors.affiliation: Department of Cancer Biology, The Scripps Research Institute, Scripps Florida, Jupiter, Florida.
authors.forename: Min
authors.initials: M
authors.lastname: Guo
authors.affiliation: Department of Tumor Biology, Moffitt Cancer Center and Research Institute, Tampa, Florida.
authors.forename: John L
authors.initials: JL
authors.lastname: Cleveland
authors.affiliation: Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina. Biomedical Research Imaging Center, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina. antonio_amelio@unc.edu.
authors.forename: Antonio L
authors.initials: AL
authors.lastname: Amelio
mesh_terms: D000818:Animals; D002051:Burkitt Lymphoma / Q000737:chemistry / Q000473:pathology; D063646:Carcinogenesis / Q000737:chemistry* / Q000473:pathology; D002289:Carcinoma, Non-Small-Cell Lung / Q000737:chemistry / Q000473:pathology; D033364:Decapoda / Q000201:enzymology; D005434:Flow Cytometry / Q000379:methods*; D049452:Green Fluorescent Proteins / Q000737:chemistry* / Q000235:genetics; D057809:HEK293 Cells; D064593:Heterografts; D006801:Humans; D008156:Luciferases / Q000737:chemistry* / Q000235:genetics; D049408:Luminescent Agents / Q000737:chemistry*; D008175:Lung Neoplasms / Q000737:chemistry / Q000473:pathology; D016688:Mice, Inbred NOD; D016513:Mice, SCID; D061848:Optical Imaging / Q000379:methods*; D011993:Recombinant Fusion Proteins / Q000138:chemical synthesis / Q000737:chemistry* / Q000235:genetics
chemical_list: D049408:Luminescent Agents; D011993:Recombinant Fusion Proteins; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D008156:Luciferases