Before, beside us, and above
The firefly lights his lamp of love.”
by Bishop Reginald Heber
Bioluminescence is one of the premier tools that scientists have in research, whether studying in vitro or in vivo. Few devices allow for the range, versatility, and ease of use as our adaption of the firefly’s twinkling star. But the firefly luminescence was only the beginning, and biologists have found many other species (mostly in shallow, coastal waters) which have developed the ability to light their own way and which we can copy for our own use and benefit.
Firefly luciferase, with its substrate luciferin, is still by far the most popular system for use in bioluminescent imaging (BLI), with Gaussia luciferase, and its substrate coelenterazine, a close second. Over the years, these two systems have been combined in various methods or kits in order to provide a more expansive research device. Most often, one or the other is used as a system control while the other pulls the heavy load. And there have been many attempts to expand the system even further, such as altering the luciferase cDNA and changing its emission spectrum in order to add a third BLI wavelength. But most of this work has been done in vitro, where the “trouble” of dealing with more than one substrate in a system can be a burden. But in vivo researchers are less bothered by such minor complications.
In animal studies, there are other things to worry about. For instance, how well does a new system handle the body temperature of the model? Can the substrate get to the test site, how quickly/slowly, and which route of injection works best? Will the substrate be broken down in the system? Can we visualize the BLI through the thousand-fold layers of cells in the animal model? There have also been many combinations of BLI and fluorescence systems in order to expand the in vivo systems as well, but with many models displaying autofluorescence, the advantages of doing so is somewhat muted by comparison. Into that line of discovery, enter Dr. Casey Maguire and his group from Harvard Medical School/Massachusetts General Hospital.
Maguire et al. wanted to develop a system in which three different luciferase signals could help report cancer cells and their cellular interactions. Firefly and Gaussia luciferases were a given, but they needed another, and decided on Vargula (or Cyprindina) luciferase. This relatively new luciferase was found in Vargula hilgendorfii (previously called Cypridina hilgendorfii), a crustacean sometimes called a sea shrimp or sea-firefly. V-Luc (or sometimes C-Luc) utilizes a substrate called Vargulin to produce a blue colored light around the 450nm wavelength. Using a mouse model, Maguire injected cancer cells intracranially which had been modified with either VLuc, FLuc or GLuc cDNA. Ultimately, they wanted to test their ability to “monitor the effect of an adeno-associated virus (AAV)-mediated soluble tumor necrosis factor-related apoptosis-inducing ligand (sTRAIL) therapy against intracranial glioma tumors.”
The results were outstanding, barring a few caveats which you can read for yourself in the discussion section of their article. There was little to no overlap in the BLI signals between the three substrates and all three were clearly visible, even in deep tissue, like the brain. The use of luciferin, coelenterazine, and now vargulin, as triple BLI reporters makes for the best of a cost-effective, sensitive and easy-to-use reporting system. And at GoldBio, that’s just the way we like it!
Maguire, Casey A., et al. "Triple Bioluminescence Imaging for In Vivo Monitoring of Cellular Processes." Molecular Therapy—Nucleic Acids 2.6 (2013): e99.
Category Code: 79101 88231