Notes: Researchers were looking for alternative methods to using transposase vectors carried by lentiviruses to insert genes into cellular DNA without the cytotoxicity that may occur if the transposase gene integrated into the genome. In this paper, the authors worked out a method to generate lentiviral particles that carried the transposase protein for delivery of genes at an equal efficiency as the conventional plasmid-based method. The reporter gene NanoLuc® luciferase was amplified from the pNL1.1[Nluc] Vector and cloned into a gag-pol-integrase-defective packaging construct. Firefly luciferase was cloned into the PB transposon lentiviral vector. Gag-pol constructs expressing the hyperactive piggyback (PB) transposase were also created. Lentiviral particles (LPs) were generated by cotransfection of several plasmids into 293T cells. One day prior to transduction, HeLa cells were seeded at a density of 10⁴ cells/well in a 96-well plate, then NanoLuc® LPs with or without pseudotyping by Vesicular Stomatitis Virus envelope glycoprotein were added. After 48 hours, luminescence was measured using the Nano-Glo® Luciferase Assay System. To analyze how well the firefly luciferase gene was transferred, HaCaT and ARPE-19 cells were seeded at 1,000 cells/well in a 96-well plate one day before transduction with increasing amounts of either wildtype or mutated PBase/firefly luciferase transposon LPs. After ten days, the transduced cells were assessed for luminescence using the ONE-Glo™ Luciferase Assay System. HEK293 cells, primary keratinocytes and normal human dermal fibroblasts were seeded at 5,000 cells/well in 24-well plates the day before transduction and then incubated with either wildtype or mutated PBase/firefly luciferase transposon LPs. After eight days, firefly luminescence was measured. (4448)

Notes: Researchers were interested in using a reporter gene that could be stably expressed in alphaviruses without attenuating infectivity. cDNA clones of eastern equine encephalitis (EEEV), Venezuelan equine encephalitis (VEEV), Sindbis (SINV) and chikungunya (CHIKV) viruses had either firefly luciferase or a FLAG-tagged NanoLuc™ luciferase genes inserted in the genomes using three different insertion points. The cDNAs were then transcribed to generate infectious viral RNAs that were then electroporated into BHK-21 cells, virus particles harvested from the supernatant after 18–24 hours and stored at –80°C in single-use aliquots as viral stock.

To assess how the reporter genes affected viral replication, BHK-21 cells were infected at a multiplicity of infection (MOI) of 0.1 PFU/cell or 5 PFU/cell. After 1 hour, cells were washed and medium replaced. At time points 0, 6, 12, 18, 24 and 48 hours, supernatant was sampled for plaque assay titration and cells lysed with 1X Passive Lysis Buffer for measuring reporter activity using the Luciferase Assay System for firefly luciferase or Nano-Glo® Luciferase Assay for NanoLuc™ luciferase.

To examine how the presence of the reporter gene might affect viral infectivity over time, BHK-21 cells were infected with SINV reporter viruses at an MOI of 0.1 PFU/cell and passage 1 (P1) viral stock was harvested 18 hours after infection. The SINV virus was then diluted 1:1,000 for infection of fresh cells, serially passaged nine more times. Supernatants from P1– P10 viruses were titrated by plaque assay; cells were lysed with 1X Passive Lysis Buffer to assay luciferase activity. Parallel protein lysates were prepared with whole-cell extract lysis buffer for Western blotting analysis using Anti-Luciferase pAb for firefly luciferase and an anti-FLAG antibody for NanoLuc™ luciferase.

Five-day-old CD-1 mice were infected with 1,000 PFU of SINV reporter viral stock in the ventral thorax region while six to eight-week-old CD-1 mice were infected with 1,000 PFU of EEEV reporter viral stock in the right rear footpad, and monitored for at least ten days. Groups of mice were sacrificed at various intervals, tissues (e.g., brain and spleen) homogenized in 1X Passive Lysis Buffer, frozen at –80°C and luciferase activity analyzed. For imaging studies, adult C57Bl/6 IFNAR1-/- mice were infected in both hind limb footpads with 1,000 PFU of either firefly or NanoLuc™ luciferase-TaV viral stocks. Six, 24 or 48 hours post-infection, 3mg of D-luciferin or 10μg of furimazine were injected into the tail vein. Mice were imaged for 2 seconds within 2 minutes of substrate administration. (4442)

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 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: To understand the mechanism of Panicle blast 1 (Pb1) gene-mediated resistance to rice blast, a rice fungal disease, researchers investigated Pb1 interacted with a transcription factor involved in resistance, WRKY45 that is regulated by the ubiquitin system. To study how these proteins interacted, inner rice leaf sheaths were bombarded with gold particles coated with 0.5 µg of effector plasmid, 0.3 µg of NanoLuc® luciferase reporter and 0.1 µg of reference Renilla luciferase. After incubating overnight at 28°C, samples were ground in liquid nitrogen and reporter activities assayed using the Dual-Glo® Luciferase Reporter Assay System and Nano-Glo® Luciferase Assay System. The Renilla luciferase gene was also split into an N-terminal construct and C-terminal construct, expressed in rice protoplasts and assayed for reconstituted Renilla luciferase activity. Expression was normalized to firefly luciferase. (4510)

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)