A secreted luciferase for ex vivo monitoring of in vivo processes

Abstract

Luciferases are widely used to monitor various biological processes. Here, we describe the naturally secreted Gaussia luciferase as a highly sensitive reporter for quantitative assessment of cells in vivo by measuring its levels in blood. The Gluc blood assay complements in vivo bioluminescence imaging which has the ability to localize the signal and provides a multifaceted assessment of cell viability, proliferation and location in experimental disease and therapy models.Luciferase-mediated bioluminescence imaging has served as a reporting tool for monitoring various biological processes in vitro and in vivo in different fields 3 , including immunology 4 oncology 5 , virology 6 , and neuroscience 7 . After systemic substrate injection, a charge coupled device (CCD) camera can be used to localize the luciferase photon signals in vivo. Recently, we have characterized a naturally secreted luciferase from the marine copepod Gaussia princeps (Gaussia luciferase, Gluc) and found it to be over 2000-fold more sensitive than firefly and Renilla reniformis luciferases and 20,000-fold more sensitive than the secreted alkaline phosphatase (SEAP) when expressed in mammalian cells 8,9 . Gluc expression levels can be easily quantified in cell-free, conditioned medium by adding its substrate coelenterazine and measuring emitted photons using a luminometer. Since Gluc is secreted from mammalian cells in culture 9 , we hypothesized that it might also be secreted into the blood of animals harboring cells expressing this reporter.In order to assess the potential of Gluc as a reporter to monitor biological processes by measuring its level in the blood of small animals, we transduced Gli36 human glioma cells with a lentivirus vector encoding Gluc (Gli36-Gluc) and implanted them in different numbers into the flanks of nude mice. We visualized the tumors 3 days post-implantation by in vivo bioluminescence imaging after intravenous (i.v.) injection of the Gluc substrate, coelenterazine (4 mg/kg body weight) and acquiring photon counts using a CCD camera (Fig. 1a). At the same time, we withdrew 5 μl blood samples from these mice and directly aliquoted them into tubes containing EDTA (1 μl 20 mM), after which we measured the Gluc activity by adding coelenterazine (100 μM) and acquiring photon counts using a luminometer (Supplementary Methods online). The Gluc activity in the blood was linear with respect to cell number in a NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript range covering over 5 orders of magnitude, and correlated well with values obtained using the CCD camera (Fig. 1b). Further, Gluc activity could also be detected in the urine, albeit to a lesser extent than in the blood, which indicates it is cleared by the kidneys (Fig. 1b). In addition, there was no detrimental effect of EDTA on Gluc activity measured in blood ( Supplementary Fig. 1a online), and no significant differences were detected between the Gluc activity measured in serum or whole blood samples, showing that hemoglobin, which can int...

Repurposing Mebendazole as a Replacement for Vincristine for the Treatment of Brain Tumors

“…Tail vein blood was collected daily and used for Gaussia luciferase quantification by luminometry assay as previously described (16). Gaussia luciferase substrate was purchased through Targeting Systems.…”

Quantitation of autophagy by luciferase release assay

“…The detection of Gaussia luciferase in whole animals can be easily accomplished using CCD-cameras. 48,77 In addition, it has been demonstrated that Gaussia luciferase secreted from tumor cells can be harvested from the blood and urine of mice 78 suggesting that the feasibility of a system that allows the detection and monitoring of autophagic pathways in whole animals by harvesting body fluids. Such a system could also be applied in animals bearing experimental tumors that have been engineered to express the sensor.…”

850 a Gaussia Luciferase Cell-Based System to Assess the Infection of Cell Culture- And Serum-Derived Hepatitis C Virus

“…Indeed, the Huh-7.5/EG(4A/4B)GLuc cell line combines the advantages of the previously reported Huh7-J20 and Huh-7.5/EG(D4B5A)SEAP cell lines while also retaining the unique features of the Gaussia reporter. These specialized features can be enumerated: (i) as Gaussia is naturally secreted into the media, no cell lysis is required for the detection of its enzymatic activity; (ii) the secreted indicator function not only permits multiple kinetics experiments using only one culture through the sequential sampling of the medium, but also allows the cells to be used for other purposes, such as RNA extraction, Western blot analysis, and other assays; (iii) Gaussia is non-toxic and very potent -compared to the humanized forms of firefly (hFLuc) and Renilla (hRLuc) luciferases expressed under similar conditions, the humanized form of Gaussia (hGLuc) generates over a 1000-fold higher bioluminescent signal intensity from live cells (in concert with their immediate environment) and over a 100-fold higher intensity from viable cells alone (not including secreted luciferase) or cell lysates [21]; (iv) Gaussia is stable in culture medium (with a half-life ,6 d) [30] and samples can thus be stored at 4uC for several days without any significant change in its activity levels; (v) it is HCV genotype-independent, and therefore does not require any genetic modification of the viral genomes, due to the highly conserved nature of the NS3/4A recognition sequence [31].…”