Abstract
Bioluminescence reporter gene imaging is a robust, high-throughput imaging modality that is useful for tracking cells and monitoring biological processes, both in cell culture and in small animals. We introduced and characterized a novel bioluminescence reporter—membrane-anchored Cypridina luciferase (maCLuc)—paired with a unique vargulin substrate. This luciferase-substrate pair has no cross-reactivity with established d-luciferin- or coelenterazine-based luciferase reporters. We compare maCLuc with several established luciferase-based reporter systems (firefly, click beetle, Renilla, and Gaussia luciferases), using both in vitro and in vivo models. We demonstrate the different imaging characteristics of these reporter systems, which allow for multiplexed-luciferase imaging of 3 and 4 separate targets concurrently in the same animal within 24 h. The imaging paradigms described here can be directly applied for simultaneous in vivo monitoring of multiple cell populations, the activity of selected signal transduction pathways, or a combination of both constitutive and inducible reporter imaging.
Highlights
Bioluminescence reporter imaging (BLI) is a frequently used imaging tool in biology and cancer research for monitoring tumor development, formation of metastases, and cell trafficking in animal models with “constitutive” reporters and for monitoring cell function and signal pathway activation with “inducible” reporters
These luciferases do not cross-react with D-luciferin or coelenterazine, nor do the conventional luciferases react with vargulin
We describe the successful development of a new BLI reporter suitable for whole body imaging—a membrane-anchored Cypridina luciferase
Summary
Bioluminescence reporter imaging (BLI) is a frequently used imaging tool in biology and cancer research for monitoring tumor development, formation of metastases, and cell trafficking in animal models with “constitutive” reporters and for monitoring cell function and signal pathway activation with “inducible” reporters. BLI is a highly robust, reliable, and sensitive imaging modality that lends itself to high throughput and is relatively low cost. It is applied throughout the field of biotechnology, from fundamental research to validation of novel clinically applicable therapeutics. A new class of bioluminescence reporters derived from Vargula hilgendorfii and Cypridina noctiluca marine ostracods[1,2] has been described, which utilize a unique substrate (vargulin) Both Vargula and Cypridina luciferase enzymes are naturally secreted from their respective hosts and catalyze oxidation of vargulin, resulting in blue light emission (465 nm). These luciferases do not cross-react with D-luciferin or coelenterazine, nor do the conventional luciferases react with vargulin
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