Notes: BDCRB selectively disrupts DNA packing in cytomegalovirus, a process which is critical for herpesvirus life-cycle. In this paper, the authors use guinea pig cytomegalovirus as a model for human infection and derive a resistant virus to characterize the BDCRB mechanism of action. Recombinant viruses containing an expression cassette for NanoLuc luciferase in either a wild-type or L406P mutant cytomegalovirus background were constructed. Luciferase activity was used as a proxy for viral yield in BDCRB treated or untreated cells. The L406P mutation did not show significant resistance to BDCRB treatment.  (5108)

Notes: Infection progression remains poorly characterized in Chagas disease, a life-long infection caused by T. cruzi. To understand the development of the disease, a bioluminescent imaging system for live mice infected with NanoLuc-labeled transgenic T. cruzi was developed. Initial expression of NanoLuc luciferase was determined in TcCOL cells and cell lysates using the Nano-Glo Live Cell assay and the Nano-Glo Luciferase Assay System, respectively. Luminescence could be detected as early as 14 days post-infection and was focused in the abdomen. Further, luminescence remained detectable for up to 222 days post infection, while parasite number in blood decreased after 21 days to the limit of detection.  (5110)

Notes: In this paper, the authors describe the development of an assay to monitor apoptosis using a novel bioluminescent method to detect annexin V binding to phosphatidylserine (PS) along with fluorescent detection of compromised membrane integrity. Assay development, characterization and benchmarking against established methods is demonstrated. (5180)

Notes: S. pneumoniae uses the ABC transporters ComAB and BlpAB in natural competence and bacterial predation through bacteriocin secretion. NanoLuc and RFluc constructs with upstream comAB and blpAB promoters were used to look at transcriptional activation. Also, bacteriocin secretion by ComAB and BlpAB was determined using the HiBiT system. BlpI was tagged with HiBiT and luciferase activity was monitored in both wild-type and mutant transporter backgrounds. Both ComAB and BlpAB were able to respond to and secrete BlpI. (5137)

Notes: Obstacles in message transfer and designer exosome production have hindered the use of exosomes as therapeutic agents. These authors describe a genetically encoded device, EXOsomal transfect into cells (EXOtic), which addresses these obstacles. A screen for genes that enhance exosome production was conducted by fusing an exosome marker to NanoLuc luciferase. Luminescence was measured using the Nano-Glo Luciferase Assay. Identified genes yielded a 15- to 40-fold increase in signal.  (5111)

Notes: A novel mechanism of tumor metastasis utilizing inflammatory monocytes (IMs) and Factor XIIIA is described. Specifically, Factor XIIIA serves as a scaffold stimulating cancer cell invasion through fibrin cross-linking. IM are recruited to tumor cells through the inflammatory chemokine, CCL2. Mice were injected with NanoLuc expressing lung squamous cancer cells and tumor progression was measured using the Nano-Glo Luciferase Assay. Treatment with a CCL2 inhibitor, preventing IM recruitment to tumor cells, displayed a significant decrease in tumor progression. (5109)

Notes: This paper details one example of using NanoLuc luciferase to create reporter viruses. NanoLuc-containing infectious particles for various types of flavivirus (e.g., Dengue, Zika, West Nile) were used in neutralization assays with serum samples following either infection or immunization. (5129)

Notes: The CL58 gene from the halotolerant green alga Chlamydomonas reinhardtii was characterized. The mRNA levels of CL58 increased in response to increased copper levels and lower temperature. Further, a NanoLuc fusion showed the expressed protein is secreted. While the function of CL58 remains unknown, here initial characterization shows it is involved in stress response.  (5112)

Notes: Phage display uses protein fusions to surface exposed membrane proteins as screening technique for protein-protein and protein-peptide interactions. Here this technique is applied for detection of imidaclothiz, a small molecule. A NanoLuc Luciferase fusion to cyclic 8-amino-acid peptidomimetic is used as a reporter for antibody detection of a imidaclothiz. Further, the authors develop two novel techniques, bioluminescent enzyme immunoassay (BLEIA) and a bioluminescence lateral flow immunoassay (BLLFIA), for use with the NanoLuc Luciferase. (5105)

Notes: Mucosal microbiota has been shown to influence HIV-1 infection. Here, P. gingivalis outer membrane vesicles (OMVs) show stimulation of receptor-independent HIV-1 entry into epithelial cells. A secretory NanoLuc luciferase is fused to the nef gene of the HIV genome, cell supernatant is collected, and luciferase activity is measured as a proxy for viral replication. The presence of P. gingivalis vesicles increased viral infectivity approximately 10-fold. (5107)

Notes: The development of NanoLuc Luciferase has led to a need for selective NanoLuc inhibitors to allow for bioluminescent suppression and multiplexing compatibility with existing assays. A lead compound with an IC50 of 600 nM against NanoLuc was further derivatized to create a family of potent and cell permeable inhibitors. These compounds were additionally developed to generate a second class of cell impermeable inhibitors. These inhibitors were tested for selectivity against Firefly luciferase and the NanoBiT system with no cross-reactivity.  (5106)

Notes: An insertional mutagenesis screen of the Dengue virus genome was used to identify regions tolerating small 15 amino acid insertions. The NS1 protein, which is essential for viral genome replication, was shown to be highly tolerant to insertions. NS1 was tagged with both a minimal fusion tag (Small BiT) and NanoLuc Luciferase. Tagged protein variants were assessed for viral infectivity, and used to investigate the protein localization and levels of NS1. (5075)

Notes: Modified antisense oligonucleotides (ASOs) show increased delivery and stability in cells, however these modifications have off-target effects. Localization of ASOs to cytoplasmic ribonucleoprotein (RNP) granules is observed to be mediated by RNA binding proteins, FUS and PSF. These interactions are further investigated using the NanoBRET™ Protein-Protein Interaction System. NanoLuc^® tagged protein is produced using an in vitro transcription and translation system, purified, and bound to an acceptor oligonucleotide (AlexaFluor594-ASO). Various backbone and 2′ ASO modifications were screened for interaction with FUS truncations to determine the interaction domain. (5061)

Notes: Proteins fused to NanoLuc^® luciferase were used as the energy donor and AlexaFluor conjugated nucleic acids were used as the energy acceptor in BRET assays that measured protein:nucleic acid interactions. The assay was demonstrated with immunopurified proteins, cell homogenates, and in whole cells. NanoLuc^® fusion protein construction, expression, and purification were performed using the vectors pFN31K Nluc CMV-neo for amino-terminal clones and pFC32K Nluc CMV-neo for carboxy-terminal clones. BRET assays were performed in white 96-well plates. Alexa-linked anti-sense oligonucleotides at the indicated concentrations were incubated at room temperature for 15 min in 1X binding buffer with 106 RLU/well of immunoprecipitated NLuc fusion protein or whole cell lysate. Following the incubation, NanoGlo^® substrate was added at 0.1 μl/well. Readings were performed using a GloMax^® Discover System. (4757)

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 rAAV-mediated genome editing technology was used to introduce a NanoLuc^® reporter sequence downstream of and in frame with the last coding exon of the HIF1A gene in HCT116 cells. This created a reporter cell line with a single allele of HIF1A endogenously tagged with a NanoLuc^® reporter fusion. The cell line was used to study compound inhibition of Hif1a signaling. NanoLuc^® luciferase activity was measured using Nano-Glo^® reagent. For qHTS, cells at 1500 cells/well in 1536-well plates were incubated with test compounds at 37°C, 5% CO₂, 1% O₂ for 18 hours in a humidified CO₂ incubator with variable oxygen control, followed by addition of Nano-Glo^® reagent or CellTiter-Glo^® cell viability assay reagent. The HRE-bla assay was conducted using the CellTiter-Glo^® reagent. HCT116 and ME-180 cell proliferation was quantified as relative luminescence unit (RLU) values using the CellTiter-Glo^® viability assay reagent. (4761)

Notes: The authors fused the PPEP-1 signal sequence to a codon-optimized luciferase gene based on NanoLuc® Luciferase (such as the pNL1.1 Vector) to create a secreted luciferase reporter for C. difficile. Measurements of luciferase activity were performed with 100µl 1:100 sample (either whole cell lysate or culture supernatant) in triplicate in a 96-well plate with 20µl NanoGlo® Luciferase Assay System. (4716)

Notes: NanoLuc® Luciferase was used to monitor Plasmodium berghei throughout it's life cycle, including detecting single parasites in mosquitos, livers and asexual blood stages. (4922)

Notes: This article details the advantages of NanoLuc® Luciferase in the scientific community. (4924)

Notes: This study used  E. coli O157:H7 bacteriophage ΦV10 modified to express NanoLuc® luciferase (Nluc) to give a detectable signal from E. coli O157:H7. (4923)

Notes: To track the fate of a microbe after it was consumed by a mosquito and migrated to the insect’s gut, a stably expressed, sensitive reporter gene that could track the bacteria with minimal effects from environmental factors was needed. The commensal bacteria Elizabethkingia anophelis was conjugated with an Escherichia coli strain carrying a plasmid with the NanoLuc® luciferase gene. Expression of NanoLuc® luciferase was measured by lysing the bacteria with Passive Lysis Buffer with lysozyme, mixing the resulting lysate with an equal volume of NanoGlo® Luciferase Assay Reagent and measuring luminescence. Larval Anopheles stephensi, Aedes triseriatus and Anopheles gambiae mosquito strains were incubated with NanoLuc® luciferase-expressing E. anophelis for 2 hours, samples taken at 0, 1, 1.5, 2 and 2.5 hours. Luciferase expression was determined by homogenizing four larvae, centrifuging the sample, washed with PBS then suspended in PBS, mixed with an equal volume of NanoGlo® Luciferase Assay Reagent and measured luminescence. Larval A. stephensi, A. triseriatus and A. gambiae mosquitos were fed NanoLuc® luciferase-carrying E. anopheles and reared to adult stage where mosquitos were homogenized and NanoLuc® luciferase expression measured. Adult A. stephensi were fed NanoLuc® luciferase-expressing E. anopheles in a 10% sucrose solution for 16–24 hours. After the 10% sucrose solution was replaced, samples of mosquitos were taken for several days and tested for NanoLuc® luciferase expression. (4573)

Notes: The authors present an overall strategy for using the CRISPR/Cas9 system for reporter tagging of endogenous loci. As examples, NanoLuc^® and TurboGFP-tagged reporter cell lines were generated. PCR was performed using GoTaq^® Polymerase. To detect the integration event, we combined a primer binding in the cassette (NanoLuc^® or TurboGFP) with a gene-specific primer. Cells were analysed using the NanoGlo^® Luciferase Assay. (4755)

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: To test the possibility of constructed constitutive circuits that would permanently activate a gene in response to a signal, researchers used a variety of combinations of the promoter for the housekeeping sigma factor from Bacteriodes thetaiotaomicron, eight ribosome binding sites (RBSs) of varying strengths, and the NanoLuc® luciferase reporter to monitor gene expression. Combinations of the promoters and RBSs produced a 10⁴-fold expression range, a range of gene expression that is comparable to commonly studied laboratory organisms. To create inducible circuits, the NanoLuc® luciferase gene was placed under the control of the rhamnose promoter such that luciferase expression was only activated in the presence of the sugar rhamnose. “Cellular memory” was added to the inducible circuit by incorporating integrases under the control of a rhamnose promoter and integrase recognition sites flanking a unique DNA sequence. In the presence of rhamnose, luciferase expression increased and produced integrase, which clipped out the unique piece DNA from the circuit, flipped it around and replaced it, recording the encounter with the signal. To inactivate genes or otherwise modify the genetic circuit, CRISPR interference (gene silencing) was used to repress NanoLuc® luciferase gene expression by activating Cas9 activity when a specific signal was received. These synthetic circuits were then tested using additional guide RNAs to knockdown endogenous genes. The engineered B. thetaiotaomicron microbes were introduced to mice and successfully colonized mouse as measured by luciferase assays of stool samples. (4572)

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)