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A variety of luciferase genes code for proteins products which catalyze the emission of light from a substrate without requiring exogenous illumination. This bioluminescence makes these genes and their proteins very useful for the noninvaseive study of biological pocesses in small laboratory animals. Using sensitive CCD cameras we can image tisses, cells or pathogens in living animals which have had these genes inserted. Although the amount of light produced is small, since rodent tissues have absolutely no natural bioluminescence properties there is no background signal allowing for highly sensitive detection.
Agar plates with bacteria expressing an orange and a yellow light emitting firefly Luciferase are shown.The bacteria colonies or streaks can be seen glowing after application of D-Luciferin substrate.
By engineering cells or specific tissues from animals with these luciferase genes, we can track the position and measure the burden of such cells in a given animal over time. The imaging procedure involves adminstration of the luciferase substrate, which can usually be given intraperitoneally; a mild anaesthetic to keep the animal still. Imaging is performed in a dark box with exposures typically under one minute. Since the procedure is very benign, it can be repeated several times in the same animal accruing valuable serial information. This system is most usually used to track the responses of experimental tumors in mice.
Mice injected with an EBV virus immortalized cell line expressing
firefly luciferase. When implanted subcutaneously, this cell line grows
and is a model for post-transplant lymphproliferative disorder.
Xenografted neuroblastoma cell lines, used as a
model for this childhood cancer.
Intravenous injection of a leukemia cell line used to model a widely disseminated cancer.
More advanced uses of bioluminescence are also possible. For instance, we have generated luciferases which emit light at different frequencies. This can allow tracking of different types of cells in the same animal. By connecting expression of a luciferase protein to a particular transcription factor, it may be possible to detect cells in animals which have differentiated into a particular lineage. Further, it is possible to engineer luciferases to emit light in response to certain intracellular molecular events by molecular recombination, or even to emit light in response to calcium fluxes.
The spectra of several engineered firefly luciferases are shown. The most extreme green enzyme has an emission peak of 550nm, while the most extreme red enzyme has an emission peak of 630. By using an appropriate filter set, it is possible to differentially image light from the red or green enzyme.
It is also possible to image small animals using fluorescence. Either tissues or cells are engineered with genes which express fluorescent proteins, or else cells and tissues are labeled with fluorescent chemicals or antibodies. In this case, the animal must be exposed to a bright excitatory light and an appropriate filter set lets the emitted light through to the camera. Problems of autoflourescence and phototoxicity must be overcome, but with spectral unmixing methodologies, it is possible to image many different fluorophores simultaneously, albeit with less sensitivity than bioluminescence. We have a wide variety of luciferases and fluorescent proteins and extensive expertise engineering both. Please feel free to contact us if you would like our collaboration or assitance to establish a fluorescence of bioluminescence based model.
Page last modified on 12 sep 11 15:00