Notes: Nanoluciferase was used to characterize the conditions required to reliably perform single-cell BRET imaging of protein-protein interaction. HEK293T cells were cultured and transfected with vectors containing the Nluc gene sequence, such as is provided in the pNL1.1 Vector. Extracellular signal-related kinase (ERK) activity in neuronal dendritic spines, induced by the activation of endogenous synaptic NMDA receptors, was detected by an Nluc-optimized BRET-based sensor of ERK activity. The authors report that the use of Nanoluciferase improved the resolution in terms of both time and space, enhanced the duration of signal stability, expanded the dynamic range and increased the sensitivity of the BRET signals in single-cell imaging. (4691)

Notes: The receptor binding assays of the mature INSL5 and R3/I5 mutants were carried out using a NanoLuc-conjugated R3/I5 peptide as tracer and the human embryonic kidney (HEK) 293T cells transiently overexpressing human RXFP3 or human RXFP4 as receptor source. The receptor activation assays of the mature INSL5 and R3/I5 mutants were carried out using a cAMP-response element (CRE)-controlled NanoLuc^® reporter. Briefly, HEK293T cells were transiently cotransfected with the expression construct of human RXFP3 or human RXFP4 and the CRE-controlled NanoLuc^® reporter vector pNL1.2/CRE. (4756)

Notes: The authors site-specifically conjugated the NanoLuc^® luciferase reporter to WT and mutant FGF2 proteins to create non-radioactive tracers that could be used to quantify ligand-receptor interactions with fibroblast growth factor receptors. Prior to Nanoluc^® conjugation, the biological activities of recombinant WT and mutant FGF2s were measured using receptor activation potency assays using a serum response element (SRE)-controlled NanoLuc^® reporter (pNL1.2/SRE) in HEK 293T cells expressing endogenous FGF receptor. Receptor-binding assays using the FGF2-Nanoluc^® tracers were carried out on overexpressed and endogenous FGF receptor in HEK293T cells. (4758)

Notes: Novel BRET “LumiFluor” reporters were created by generating fusions of NanoLuc^® luciferase with eGFP or LSSmOrange fluorescent proteins. Intramolecular energy transfer from NanoLuc^® and the fused fluorophore generated a reporter that provided a bright signal with spectral properties compatible with in vivo imaging methods. Cells expressing the LumiFluor reporters were used for xenografts, bioluminescent imaging and flow cytometry. (4759)

Notes: The authors were interested in creating a genetic manipulation system to screen drugs for inhibiting or killing Cryptosporidium parvum. Sprorozoities were excysted from Cryptosporidium oocysts purified from infected calf feces that passed through an environment that mimicked stomach and intestinal passage. These sporozoites were then transfected using electroporation with plasmids carrying reporter genes including NanoLuc® luciferase flanked by C. parvum 5´ and 3´′ regulatory sequences from highly expressed genes. Then the transfected sporozoites infected human ileocaecal adenocarcinoma cells (HCT-8) and reporter activity measured after 48 hours. Of the reporter genes tested, only NanoLuc® Luciferase (Nluc) was able to be detected. Nluc was fused to neomycin and to create a Nluc repair assay, a termination mutant Dead Nluc was created. Sporozoites were transfected with either Dead Nluc alone or Dead Nluc plus Cas9 and specific guide RNA (gDNA), and luciferase activity measured. The Cas9 with gDNA restored Nluc activity. To further test this system and see if it could disrupt a gene associated with drug resistance, Nluc-Neo with Cas9 and gDNA targeted thymidine kinase. With the target gene removed, the parasite was more susceptible to antifolate trimethoprim. (4571)

Notes: Researchers were interested in quantitating the ingestion of Flavobacterium hibernum by eastern tree hole mosquito (Aedes triseriatus) larvae, and better understand the fate of the bacteria once ingested. The NanoLuc™ luciferase gene Nluc was inserted into a bacterial expression plasmid then introduced to F. hibernum by conjugation to create a bacterial strain expressing NanoLuc™ luciferase. The researchers determined the number of bacteria by diluting cultured F. hibernum, adding an equal volume of Nano-Glo® Luciferase Assay Reagent and measuring luminescence. Alternatively, the bacteria were lysed with Passive Lysis Buffer and lysozyme before assaying with an equal volume of Nano-Glo® Luciferase Assay Reagent. This method could determine the number of bacteria down to 700 cells/ml. To study the larval ingestion of the NanoLuc™-expressing F. hibernum, the bacteria were cultured, washed in PBS and resuspended to 7.3 × 10⁵ cells/ml. Three milliliters of this feeding solution was inoculated with six A. triseriatus larvae of three different stages [3rd instar larvae (6 days past hatch), 4th instar larvae (9 days past hatch) and pupae (12 days past hatch)]. The number of bacterial cells remaining after a feeding interval was sampled and quantitated using the Nano-Glo® reagent as detailed earlier.
Third-instar larvae were starved for 2 hours in sterile water then 2.8 × 10⁹ cells/ml of the NanoLuc™ expression bacteria were added and incubated with the larvae for 4 hours. After incubation, the mosquito larvae were rinsed well, and 2ml of larvae suspended in sterile water were transferred to a six-well plate to assess the rate of digestion. The amount of bacteria inside the larvae and the incubation solution were determined by sampling at 0, 0.5, 1, 1.5, 2, 2.5, 3 and 4 hours. To quantitate the internal bacteria, three larvae were pooled, homogenized, re-suspended in water and analyzed using the Nano-Glo® Luciferase Assay Reagent. To more closely mimic the tree hole environment in which the A. triseriatus larvae feed, six 2nd instar larvae were transferred to a 20ml microcosm of tree-hole water and beech leaf that was then inoculated with the NanoLuc™-expressing F. hibernum at 4.7 × 10⁵ cells/ml. Bacterial cells in the microcosm were sampled at 0, 1, 2, 3 and 6 days, washed, resuspended in PBS and 50µl used to measure NanoLuc™ luciferase activity. (4441)

Notes: Hepcidin is a small peptide secreted by the liver that plays a key role in iron homeostasis by binding and internalizing the iron efflux transporter ferroportin (Fpn). The authors of this paper used NanoLuc® luciferase-tagged and GFP-tagged Hepcidin fusion proteins to measure the internalization of Fpn in HEK293T cells. Once the NanoLuc®-tagged Fpn was internalized, luminescence was significantly decreased. The authors coexpressed both NanoLuc®-tagged Fpn and GFP-tagged Fpn using a doxycycline-inducible bidirectional promoter and were able to measure hepcidin-induced Fpn internalization qualitatively (GFP fluorescence) and quantitatively (bioluminescence).  (4436)

Notes: In vivo imaging using bioluminescent reporters is a powerful tool for real-time detection of viral load and spread in an animal over time. However, construction of influenza reporter viruses is complicated because the small viral genome does not tolerate large insertions and all the viral genes are critical in vivo, making it impossible to replace any gene with a reporter. These authors describe construction of a replication-competent influenza reporter virus containing the small (19kDa), bright NanoLuc® luciferase gene. NanoLuc® luciferase activity was then used to monitor viral infection in real time in an animal model.  Bioluminescent imaging of the reporter virus allowed serial observations of viral load and dissemination in infected animals, even following clearance of a sublethal challenge. (4435)

Notes: These authors demonstrated use of NanoLuc® Luciferase bioluminescence for imaging applications in mice. They showed that bioluminescence could be imaged in superficial and deep tissues, and were able to monitor changes in bioluminescence over time to quantify tumor growth. Secreted NanoLuc® Luciferase was also detectable in small volumes of serum. The paper also details use of both NanoLuc® and firefly luciferase reporters in a dual assay to quantify two steps in TGFβ signaling both in intact cells and in living mice. (4439)

Notes: These authors analyzed the performance of the following reporter enzymes used to measure biological pathway modulation by small molecules: firefly luciferase, Renilla reniformis luciferase, β-lactamase, mutated forms of Renilla luciferase emitting either blue- or green-shifted luminescence, a red-light emitting form of Luciola cruciata firefly luciferase, a mutated form of Gaussia princeps luciferase, and NanoLuc® luciferase. They screened a collection of more than 42,000 PubChem compounds using purified enzyme preparations to determine hit rates and then examined structure:activity relationships. The study evaluated hit rates and inhibitor overlap between reporters. Based on these results, the authors suggest strategies to improve the construction and interpretation of assays using these reporter enzymes. (4437)

Notes: These authors developed a sensitive receptor-binding assay for detection of interactions between the peptide hormone Insulin-like peptide-3 (INSL3) and the relaxin family peptide receptor RXFP2.  Recombinant INSL3 was tagged with NanoLuc® Luciferase by chemical modification of INSL3 to include an active disufide bond, and engineering of a 6× His-Cys-NanoLuc with an exposed N-terminal cysteine. The NanoLuc®-conjugated INSL3 was used to monitor the receptor-binding of a variety of ligands. (4438)

Notes: These authors describe the engineering of an enzyme and substrate to create a novel highly efficient bioluminescence system. The paper introduces NanoLuc™ Luciferase, a small luciferase subunit (19kDa) from the deep-sea shrimp Oplophorus gracilirostris, which provides ∼2.5 millionfold improved luminescence activity in mammalian cells by merging optimization of protein structure with development of a novel imidazopyrazinone substrate (furimazine). The glow-type luminescence (signal half-life >2 hours) produced by the new luciferase has a specific activity ∼150-fold greater than either firefly or Renilla luciferases similarly configured for glow-type assays. In mammalian cells, NanoLuc™ Luciferase shows no evidence of post-translational modifications or subcellular partitioning. The enzyme exhibits high physical stability, retaining activity with incubation up to 55°C or in culture medium for >15 hours at 37°C.
The authors discuss utility of NanoLuc™ Luciferase as a genetic reporter configured for high sensitivity or for response dynamics through addition of a degradation sequence to reduce intracellular accumulation. Data shows the effect of adding a signal sequence to allow export of NanoLuc™ Luciferase to the culture medium, allowing measurement of enzyme activity without cell lysis. (4295)