1.1. Detection of Red-Shifted Luciferase

**Some Cellecta CloneTracker XP libraries incorporate a red-shifted luciferase (rLuc) derived from Photinus pyralis (Ppy RE9 mutant) as a reporter for bioluminescent imaging (BLI). The CloneTracker XP libraries with the Ppy RE9 rLuc were designed for this purpose. Luciferase reporters provide a useful tool for indirect cell labelling and tracking of cell fates in vivo. While the BLI technique has been successfully used for monitoring of migration, growth, and viability associated with drug treatment of transplanted cancer, stem, or immune cells in mouse models, standard luciferase assays in animal models are particularly sensitive because they are not subject to high background as a result of tissue autofluorescence. Far-red emitting enzymes offer enhanced sensitivity and resolution in deep tissue.

CloneTracker XP barcoded libraries with rLuc employ a highly sensitive, codon-optimized rLuc that emits 50-100-fold better light intensity than the click beetle (Renilla) enzyme. Cancer cells transduced with CloneTracker XP libraries that have the rLuc reporter can be used for monitoring tumor and metastasis growth in drug-treated mouse models. BLI of rLuc-tagged cells in vivo requires the use of an appropriate imager for detecting photons generated from D-Luciferin substrate by CCD cameras able to sample the entire visible and near infrared spectrum. For details on in vivo imaging, please refer to the standard protocols provided with your instrument, for example, In vivo Bioluminescence Imaging of Luciferase-labeled Cancer Cells protocol for BLI using the IVIS Spectrum imager from PerkinElmer.

Additional information, protocols, and applications can also be found in the following references:

Branchini BR, Ablamsky DM, et al. Red-emmiting luciferases for bioluminescence reporter and imaging applications. Anal. Biochem. (2010) 396:290-297.
 
Kim JE, Kalimthu S, Ahn BC. In vivo cell tracking with bioluminescense imaging. Nucl. Med. Mol. Imaging (2015) 49:3-10.
 
Kosher B, Piwnica-Worms D. Illuminating cancer systems with genetically engineered mouse models and coupled luciferase reporters in vivo. Cancer Discov. (2013) 3:616-629.