Optical imaging of Renilla luciferase reporter gene expression in living mice

Abstract

Imaging reporter gene expression in living subjects is a rapidly evolving area of molecular imaging research. Studies have validated the use of reporter genes with positron emission tomography (PET), single photon emission computed tomography (SPECT), MRI, fluorescence with wild-type and mutants of green fluorescent protein, as well as bioluminescence using Firefly luciferase enzyme͞protein (FL). In the current study, we validate for the first time the ability to image bioluminescence from Renilla luciferase enzyme͞protein (RL) by injecting the substrate coelenterazine in living mice. A highly sensitive cooled charge-coupled device camera provides images within a few minutes of photon counting. Cells, transiently expressing the Rluc were imaged while located in the peritoneum, s.c. layer, as well as in the liver and lungs of living mice tail-vein injected with coelenterazine. Furthermore, D-luciferin (a substrate for FL) does not serve as a substrate for RL, and coelenterazine does not serve as a substrate for FL either in cell culture or in living mice. We also show that both Rluc and Fluc expression can be imaged in the same living mouse and that the kinetics of light production are distinct. The approaches validated will have direct applications to various studies where two molecular events need to be tracked, including cell trafficking of two cell populations, two gene therapy vectors, and indirect monitoring of two endogenous genes through the use of two reporter genes.

Noninvasive optical imaging of nitroreductase gene-directed enzyme prodrug therapy system in living animals

“…In addition, there are difference in animal batches, clonal selections, transfection efficiency and levels of gene expression in different cells. It was interesting to note that unlike bioluminescent signals, 31 which tapered within 2 h, the signal in these NTR tumors often persisted overnight. Such unique capabilities can open up many new applications in the areas of gene imaging and cell tracking.…”

Optical imaging of transferrin targeted PEI/DNA complexes in living subjects

“…[13][14][15][16][17]19,20 Firefly luciferase gene expression has been monitored in transfected cell implants 19,20 and after delivery by viral vectors. 14,16 Recently, Iyer et al 18 have shown the first example of bioluminescent imaging of liposomal gene delivery, but polycationic gene delivery using a targeted vector complex has not been documented.…”

“…We are also validating more sensitive optical repoter genes such as a synthetic renilla luciferase, which should lead to a log order more sensitivity. 20 With continued improvement in sensitivity of detection, formulations with very low efficiency should be able to be utilized with noninvasive optical CCD imaging. This is the first study to directly compare these PEI formulations.…”

Molecular imaging of gene therapy for cancer

“…The luciferases from Renilla and firefly have different substrates, coelenterazine and D-luciferin, respectively, and can be imaged in tumors in the same living mouse with kinetics of light production being separable in time by separate injections of these two substrates. 88 Recently, dual bioluminescence imaging has been used to monitor gene delivery via a therapeutic vector and to follow the effects of the therapeutic protein TRAIL (tumor necrosis factor-related apoptosis-inducing ligand) in gliomas. 70 Glioma cells stably expressing Fluc were implanted subcutaneously into nude mice and the tumor growth was monitored in vivo over time by luciferin administration and BLI.…”