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.
Highlights
During the last decade, bioluminescent (BL) imaging has become an indispensable tool for visualizing molecular events at a cellular level both in vivo and in vitro leading to new advances and discoveries in life sciences [1].There are many available BL luciferase/luciferin reporter gene systems for in vivo imaging:the first reportedly used, and most popular, are the luciferases that require D-luciferin and are ATP dependent [2].Other luciferases followed such as Renilla luciferase and Gaussia luciferase which require coelenterazine as a substrate and are ATP independent [2,3]
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
We evaluated for the first time a new red-codon optimized luciferase, Ppy RE8, in combination with a green click beetle, CBG99, luciferase that permitted a simultaneous, sensitive and reliable 2D imaging and quantification of different imaging signals in vivo using the same D-luciferin substrate
Summary
Bioluminescent (BL) imaging has become an indispensable tool for visualizing molecular events at a cellular level both in vivo and in vitro leading to new advances and discoveries in life sciences [1].There are many available BL luciferase/luciferin reporter gene systems for in vivo imaging:the first reportedly used, and most popular, are the luciferases that require D-luciferin and are ATP dependent (i.e. firefly luciferase, click beetle luciferase) [2].Other luciferases followed such as Renilla luciferase and Gaussia luciferase which require coelenterazine as a substrate and are ATP independent [2,3]. Renilla and Gaussia luciferases emit blue light which in part compromise their in vivo performance due to extensive light absorption by the small animal body. Despite 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 colorcoupled 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
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