Sensitive Dual Color In Vivo Bioluminescence Imaging Using a New Red Codon Optimized Firefly Luciferase and a Green Click Beetle Luciferase

Abstract

BackgroundDespite a plethora of bioluminescent reporter genes being cloned and used for cell assays and molecular imaging purposes, the simultaneous monitoring of multiple events in small animals is still challenging. This is partly attributable to the lack of optimization of cell reporter gene expression as well as too much spectral overlap of the color-coupled reporter genes. A new red emitting codon-optimized luciferase reporter gene mutant of Photinus pyralis, Ppy RE8, has been developed and used in combination with the green click beetle luciferase, CBG99.Principal FindingsHuman embryonic kidney cells (HEK293) were transfected with vectors that expressed red Ppy RE8 and green CBG99 luciferases. Populations of red and green emitting cells were mixed in different ratios. After addition of the shared single substrate, D-luciferin, bioluminescent (BL) signals were imaged with an ultrasensitive cooled CCD camera using a series of band pass filters (20 nm). Spectral unmixing algorithms were applied to the images where good separation of signals was observed. Furthermore, HEK293 cells that expressed the two luciferases were injected at different depth in the animals. Spectrally-separate images and quantification of the dual BL signals in a mixed population of cells was achieved when cells were either injected subcutaneously or directly into the prostate.SignificanceWe report here the re-engineering of different luciferase genes for in vitro and in vivo dual color imaging applications to address the technical issues of using dual luciferases for imaging. In respect to previously used dual assays, our study demonstrated enhanced sensitivity combined with spatially separate BL spectral emissions using a suitable spectral unmixing algorithm. This new D-luciferin-dependent reporter gene couplet opens up the possibility in the future for more accurate quantitative gene expression studies in vivo by simultaneously monitoring two events in real time.

Evaluation of immunogen delivery by DNA immunization using non-invasive bioluminescence imaging

“…The luminescent light it emits is detectable even after passing through tissue. 12,13 This makes the signal traceable even when its origin is in the tissues beyond the skin, which allows for the use of more sophisticated methods of reporter monitoring such as bioluminescence tomography (BLT). While two-dimensional (2D) imaging allows for a rapid screening of protein expression, BLT provides detailed three-dimensional (3D) information about the anatomical location of the signal.…”

Dual-Color Bioluminescence Imaging for Simultaneous Monitoring of the Intestinal Persistence of Lactobacillus plantarum and Lactococcus lactis in Living Mice

“…Similar conclusions also have been made by Mezzanotte et al on imaging the liver and prostate using a red-optimized Fluc with CBGluc (9). The lungs, the liver, the prostate, and the GIT are difficult sites to image optically due to the great tissue depth, and the use of redemitting luciferases is more adapted to imaging in these tissues, as the longer wavelength enables better penetration through living tissues (4,(8)(9)(10).…”

“…Dual-color bioluminescence has been described with red and green luciferases expressed by mammalian cells that can be separated in vivo (9). Very recently, Chang et al showed that Fluc and CBRluc, which emit distinct light wavelengths, allow simultaneous imaging of two mycobacterial strains during infection (10).…”

In Vivo Cell Tracking with Bioluminescence Imaging

“…BLI can enable simultaneous visualization of monitoring for the expression of two divergent luciferase proteins by use of their specific substrates, i.e., the dissimilarity in the substrates for each luciferase makes it possible to selectively distinguish between the luminescent reactions for each enzyme. The imaging dual luciferase gene (red codon optimized firefly luciferase and a green click beetle luciferase) activities in the same animals could reduce variations from individual differences of the experimental animals [52]. This type of experimental design is particularly useful when trying to acquire accurate, quantitative data on gene expression by simultaneously monitoring two events in real time.…”