Abstract
Fluorescence and bioluminescence imaging have different advantages and disadvantages depending on the application. Bioluminescence imaging is now the most sensitive optical technique for tracking cells, promoter activity studies, or for longitudinal in vivo preclinical studies. Far-red and near-infrared fluorescence imaging have the advantage of being suitable for both ex vivo and in vivo analysis and have translational potential, thanks to the availability of very sensitive imaging instrumentation. Here, we report the development and validation of a new luciferase fusion reporter generated by the fusion of the firefly luciferase Luc2 to the far-red fluorescent protein TurboFP635 by a 14-amino acid linker peptide. Expression of the fusion protein, named TurboLuc, was analyzed in human embryonic kidney cells, (HEK)-293 cells, via Western blot analysis, fluorescence microscopy, and in vivo optical imaging. The created fusion protein maintained the characteristics of the original bioluminescent and fluorescent protein and showed no toxicity when expressed in living cells. To assess the sensitivity of the reporter for in vivo imaging, transfected cells were subcutaneously injected in animals. Detection limits of cells were 5 × 103 and 5 × 104 cells for bioluminescent and fluorescent imaging, respectively. In addition, hydrodynamics-based in vivo gene delivery using a minicircle vector expressing TurboLuc allowed for the analysis of luminescent signals over time in deep tissue. Bioluminescence could be monitored for over 30 days in the liver of animals. In conclusion, TurboLuc combines the advantages of both bioluminescence and fluorescence and allows for highly sensitive optical imaging ranging from single-cell analysis to in vivo whole-body bioluminescence imaging. FigOptical imaging using TurboLuc fusion reporter protein
Highlights
From its early development in the 1990s, in vivo optical imaging (OI) demonstrated a growing interest by the scientific community and a large number of applications in many research areas from plant research to biochemical, biopharmaceutical, and biomedical sciences [1]
Whole protein extracts derived from cells transfected with pTurboLuc revealed the presence of an 88-kDa protein band as a result of the fusion of Luc2 protein (62 kDa) and TurboFP635 (26 kDa)
Immunofluorescence staining on transfected cells using an antiluciferase antibody confirmed the correct expression of transfected with an equal amount of pTurboLuc vector or the parental pTurboFP635, and fluorescence emission was analyzed 24 h later
Summary
From its early development in the 1990s, in vivo optical imaging (OI) demonstrated a growing interest by the scientific community and a large number of applications in many research areas from plant research to biochemical, biopharmaceutical, and biomedical sciences [1]. Optical reporter genes have been modified to ameliorate luminescent properties of the proteins like the photo- and thermostability, maturation time, emission spectra, and quantum yields This resulted in improved in vivo sensitivity and stability characteristics of optical reporters that enabled multiple readouts and (development of) highthroughput assays. The high photostability offers advantages for stimulated emission depletion (STED) microscopy by which super-resolution is achieved in studies to elucidate protein structure analysis at sub-organelle level. These unique characteristics make TurboFP635 an attractive component for biosensors in cells, for deep tissue imaging, and long-term intravital imaging [10]. For whole-body optical imaging in small laboratory animals, TurboFP635 has shown to be superior to other reported FPs [11]
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