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Nat Chem. Biol. 12(12), 1097–1104. Potent and selective bivalent inhibitors of BET bromodomains. 2016

Waring, M.J., Chen, H., Rabow, A.A., Walker, G., Bobby, R., Boiko, S., Bradbury, R.H., Callis, R., Clark, E., Dale, I., Daniels, D.L., Dulak, A., Flavell, L., Holdgate, G., Jowitt, T.A., Kikhney, A., McAlister, M., Méndez, J., Ogg, D., Patel, J., Petteruti, P., Robb, G.R., Robers, M.B., Saif, S., Stratton, N., Svergun, D.I., Wang, W., Whittaker, D., Wilson, D.M. and Yao, Y.

Notes: The bromodomain and extraterminal (BET) family of proteins contain two bromodomains. A probe compound, biBET, capable of binding both bromodomains of BET proteins in cis is characterized. BDR4-NanoLuc and Halo-tagged histone H3 fusions are used to monitor biBET binding with the NanoBRET Target Engagement system. Interestingly, bivalent binding lead to slower displacement of inhibitor from BDR4 and increased potency. (5078)

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ACS Synth. Biol. 5, 1376–1382. The signal sequence of the abundant extracellular metalloprotease PPEP-1 can be used to secrete synthetic reporter proteins in Clostridium difficile. 2016

Oliveira Paiva, A.M., Friggen, A.H., Hossein-Javaheri, S. and Smits, W.K.

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)

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Cell Rep. 16, 37–47. The TIP60 complex is a conserved coactivator of HIF1A. 2016

Perez-Perri, J.I., Dengler, V.L., Audetat, K.A., Pandey, A., Bonner, E.A., Urh, M., Mendez, J., Daniels, D.L., Wappner, P., Galbraith M.D. and Espinosa, J.M.

Notes: HaloTag® Pull-Down Assay
HEK293T (12 × 106 cells) were plated and grown to 70–80% confluence (approximately 18 hours). The cells were then transfected (using FuGENE® HD Transfection Reagent [Cat.# E2311]) with either 30µg of HaloTag(HT)-HIF1A or HT-alone control vector (vectors available by custom order from Promega Custom Assay Services). Clarified lysates from both HT-HIF1A and HT-alone control cells were prepared and incubated with HaloLink™ Resin (HaloTag® Mammalian Pull-Down System [Cat.# G6500, G6504]). Proteins were digested with trypsin, and digestion was quenched with formic acid. Digested peptides were analyzed by mass spectrometry.

NanoBRET™ Assay
HCT116 and HEK293 cells (8 ×105) were plated in each well of a 6-well plate and co-transfected with one of three acceptors: HT-Pontin, HT-Reptin or HT-TIP60, in combination with the HIF1A-NanoLuc(NL) donor. The following NanoBRET pairs used are available by custom order from Promega Custom Assay Services: HIF1α-NLuc + HT-TIP60, HIF1α-NLuc + HT-Pontin or HIF1α-NLuc + HT-Reptin. (4718)

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Sci. Rep. 6, 33235. The Use of a Novel NanoLuc -Based Reporter Phage for the Detection of Escherichia coli O157:H7 2016

Zhang, D., Coronel-Aguilera, C.P., Romero, P.L., Perry, L., Minocha, U., Rosenfield, C., Gehring, A.G., Paoli, G.C., Bhunia, A.K. and Applegate, B.

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)

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J. Med. Chem. 58, 2718–36. 9H-Purine Scaffold Reveals Induced-Fit Pocket Plasticity of the BRD9 Bromodomain. 2015

Picaud, S., Strocchia, M., Terracciano, S., Lauro, G., Mendez, J., Daniels, D.L., Riccio, R., Bifulco, G., Bruno, I. and Filippakopoulos, P.

Notes: The authors used bioluminescence resonance energy transfer (BRET) to test the ability of a bromodomain 9 ligand to disrupt binding to histone. HEK 293 cells were cotransfected with a histone H3.3-HaloTag® fusion vector and either NanoLuc®-BRD9 bromodomain or NanoLuc®-full-length BRD4 fusion vector. After 24 hours, the transfected cells were trypsinized, diluted in phenol red-free DMEM with or without 10nM of HaloTag® NanoBRET™ 618 Ligand and dispensed into a 96-well plate. One of two potential BRD-disrupting compounds, 7d or 11, was adding to a final concentration of 0.005–33μM, cells were incubated for 18 hours and NanoBRET™ Nano-Glo® Substrate (final concentration 10µM) was added. Fluorescence was measured and a corrected BRET ratio calculated. Cytotoxicity was assessed after the NanoBRET™ assay by incubating the cells with the CellTiter-Glo® Reagent for 30 minutes and measuring luminescence. To examine histone H3.3 localization, HEK 293 cells were transfected with the histone H3.3-HaloTag® fusion vector using FuGENE® HD Transfection Reagent. After 24 hours, cells were labeled with 5μM HaloTag® TMR ligand for 15 minutes before washing with complete medium, incubated for 30 minutes and imaged with a confocal microscope. (4568)

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Nat. Commun. 17, 10237. A generic strategy for CRISPR-Cas9-mediated gene tagging 2015

Lackner, D.H., Carré, A., Guzzardo, P.M., Banning, C., Mangena, R., Henley, T., Oberndorfer, S., Gapp, B.V., Nijman, S.M., Brummelkamp, T.R. and Bürckstümmer, T.

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)

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Anal. Biochem. 489, 1–8. A luminescent assay for real-time measurements of receptor endocytosis in living cells. 2015

Robers, M. B., Binkowski, B. F., Cong, M., Zimprich, C., Corona, C., McDougall, M., Otto, G., Eggers, C. T., Hartnett, J., Machleidt, T., Fan, F. and Wood, K. V.

Notes: The authors describe a new method for real-time analysis of ligand-mediated receptor endocytosis in living cells. They used a BRET-based assay to detect the interactions of ligands with various GPCRs fused to NanoLuc® luciferase. (4693)

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Nature Methods 12(7), 661–663. Application of BRET to monitor ligand binding to GPCRs. 2015

Stoddart, L.A., Johnstone, E.K.M., Wheal, A.J.,  Goulding, J., Robers, M.B., Machleidt, T., Wood, K.V., Hill, S.J., and Pfleger, K.D.G.


Notes: These authors describe a new method for monitoring ligand binding to GPCRs on the surface of living cells. They used NanoLuc luciferase in a BRET-based assay to detect the interactions of ligands with the receptors with an N-terminal NanoLuc fusion. (4589)

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ACS Chemical Biology 10, 1188–97. Chemogenomic Profiling of Endogenous PARK2 Expression Using a Genome-Edited Coincidence Reporter. 2015


Hasson, S.A., Fogel, A.I., Wang, C., MacArthur, R., Guha, R., Heman-Ackah, S., Martin, S., Youle, R.J,, and Inglese, J.

Notes: These authors describe use of firefly and NanoLuc® luciferases in a coincidence reporter system to screen compounds for their ability to activate transcription of the Parkin gene (PARK2), a potential target for treatment of Parkinson's disease. In a coincidence reporter system, a single transcript containing two luciferase genes is generated. Inclusion of a 2A “ribosomal skipping” sequence between the two luciferase genes enables translation of two reporter proteins. This approach is useful in drug screening because the use of two independent reporters to monitor a single transcriptional event provides a way to distinguish true “hits” from experimental artifacts caused by interaction between library compounds and reporter proteins. True “hits” (compounds that affect transcription of the gene being monitored) cause a signal from both reporters; false positives caused by interaction of compounds with the reporter protein cause a signal from only one reporter. This paper describes the first use of firefly and NanoLuc® luciferases, and the Nano-Glo® Dual-Luciferase® Reporter Assay, in a coincidence reporter system to screen a compound library in an HTS assay. (4565)

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Sci. Signal. 8 (405), ra123. DOI: 10.1126/scisignal.aab4068. Distinct profiles of functional discrimination among G proteins determine the actions of G protein–coupled receptors. 2015

Masuho, I., Ostrovskaya, O., Kramer, G.M.,  Jones, C.D., Xie, K., and Martemyanov, K.A.

Notes: These authors studied the interactions between G-protein coupled receptors (GPCRs)  and 13 different G-proteins using a NanoBRET assay. NanoBRET assays were performed using protein partners labeled with NanoLuc luciferase or Venus yellow fluorescent protein. The improvement in signal-to-noise ratio achieved using the NanoBRET method enabled resolution of signals from previously intractable G-proteins and GPCRs. The authors demonstrated that GPCRs engage multiple G-proteins with distinct patterns of activity or “fingerprints”. This differential engagement of multiple target G-proteins was revealed by quantitative analysis of G-protein activation kinetics.  (4588)

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Appl. Environ. Microbiol. 81, 2233–43. Elizabethkingia anophelis: Molecular manipulation and interactions with mosquito hosts. 2015

Chen, S., Bagdasarian, M and Walker, E.D.

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)

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Cancer Res. 75, 5023-5033. Fluorophore-NanoLuc BRET Reporters Enable Sensitive In Vivo Optical Imaging and Flow Cytometry for Monitoring Tumorigenesis 2015

Schaub, F.X., Reza, M.S., Flaveny, C.A., Li, W., Musicant, A.M., Hoxha, S., Guo, M., Cleveland, J.L. and Amelio, A.L.

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)

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Nature 523, 477–480. Genetic modification of the diarrhoeal pathogen Cryptosporidium parvum. 2015

Vinayak, S., Pawlowic, M.C., Sateriale, A., Brooks, C.F., Studstill, C.J., Bar-Peled, Y., Cipriano, M.J. and Striepen, B.

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)

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Angewandte Chemie International Edition 54, 6217–21. LP99: Discovery and Synthesis of the First Selective BRD7/9 Bromodomain Inhibitor 2015

Clark, P.G.K., Vieira, L.C.C., Tallant, C., Fedorov, O., Singleton, D.C., Rogers, C.M., Monteiro, O.P., Bennett, J.M., Baronio, R., Müller, S., Daniels, D.L., Méndez, J., Knapp, S., Brennan, P.E. and Dixon, D.J.

Notes: To characterize the effectiveness of LP99, a potential bromodomain inhibitor, BRD7 and BRD9 were fused with NanoLuc® luciferase and histones H3.3 and H4 were fused with HaloTag® protein for use in BRET. The two proteins were expressed in HEK 293 cells, and the histone-HaloTag® fusions were fluorescently labeled with the HaloTag® NanoBRET™ 618 Ligand. Once the NanoBRET™ Nano-Glo® Substrate was added, NanoBRET™ ratios were assessed in the presence of varying concentrations of LP99. (4567)

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Mol. Endocrinol. 29, 1037-1054. miR-22 and miR-29a Are Members of the Androgen Receptor Cistrome Modulating LAMC1 and Mcl-1 in Prostate Cancer 2015

Pasqualini, L., Bu, H., Puhr, M., Narisu, N., Rainer, J., Schlick, B., Schäfer, G., Angelova, M., Trajanoski, Z., Börno, S.T., Schweiger, M.R., Fuchsberger, C. and Klocker, H.

Notes: The authors investigated the interaction between androgen receptors and miRNAs in order to better understand the role of miRNAs in prostate cancer biology. A NanoLuc® and firefly luciferase miRNA target expression vector (pmirNanoGlo) was used to evaluate the predicted role of miR-22 and miR-29a in the regulation of target genes LAMC1 and MCL1. This bicistronic vector contains NanoLuc® luciferase (NlucP) as the primary reporter gene and Firefly luciferase (Luc2) as the control reporter for normalization. For each target gene, the cDNA fragment predicted to encompass miR-22 and miR-29a target sites, was inserted into multiple cloning regions located in the 3′-UTR of the NanoLuc® reporter vector. PC3 cells were transfected with the reporter vectors in combination with either miR-22 mimic, miR-29a mimic and the Negative Control 4 or antimiR-22, antimiR-29a, and the Negative Control. Transfected PC3 cells underwent NanoLuc® and Firefly luciferase activity measurements using the NanoGlo® Dual-Luciferase® Assay. (4753)

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ACS Chemical Biology 10, 1797–1804. NanoBRET—A Novel BRET Platform for the Analysis of Protein–Protein Interactions. 2015

Machleidt, T, Woodroofe, C.C., Schwinn, M.K., Méndez, J.,  Robers, M.B., Zimmerman, K., Otto, P., Daniels, D.L., Kirkland, T.A., and Wood, K.V.

Notes: This paper introduces NanoBRET technology, which provides an improved alternative to conventional BRET protein interaction assays. NanoBRET assays combine the extremely bright NanoLuc luciferase with a means for tagging intracellular proteins with a long-wavelength fluorophore (HaloTag). The greater light intensity and improved spectral resolution of the NanoBRET assay results in increased detection sensitivity and dynamic range over current BRET technologies. Performance of the assay is demonstrated using several model systems, and the ability to image BRET in individual cells is illustrated. The  authors also demonstrate the application of NanoBRET in a novel assay developed for analyzing the interactions of bromodomain proteins with chromatin in living cells. (4575)

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Cell Systems 1, 62–71. Programming a human commensal bacterium, Bacteroides thetaiotaomicron, to sense and respond to stimuli in the murine gut microbiota 2015

Mimee, M., Tucker, A.C., Voigt, C.A. and Lu, T.K.

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)

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Nat. Commun. 6, 10091 doi:10.1038/ncomms10091. Target engagement and drug residence time can be observed in living cells with BRET. 2015

Robers, M.B, Dart, M.L., Woodroofe, C.C,  Zimprich, C.A., Kirkland, T.A., Machleidt, T., Kupcho, K.R., Levin, S., Hartnett, J.R., Zimmerman, K., Niles, A.L., Ohana, R.F., Daniels, D.L., Slater, M., Wood, M.G., Cong, M., Cheng Y., and Wood, K.V.


Notes: This paper describes a method for using bioluminescence resonance energy transfer (BRET) to reveal the binding characteristics of a drug with selected targets in live cells. The authors used cell-permeable fluorescent tracers in a competitive binding assay to quantify drug engagement with target proteins fused to Nanoluc luciferase. Using this approach, they were able to profile isozyme-specific engagement and binding kinetics for a panel of histone deacetylase (HDAC) inhibitors. (4587)

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ACS Med. Chem. Lett. 5, 1190–1195. 1,3-Dimethyl Benzimidazolones Are Potent, Selective Inhibitors of the BRPF1 Bromodomain. 2014


Demont, E.H., Bamborough,P., Chung,C., Craggs, P.D., Fallon, D., Gordon, L.J., Grandi, P., Hobbs, C.I., Hussain, J., Jones, E.J., Le  A., Michon, A., Mitchell, D.J., Prinjha, R.K., Roberts, A.D., Sheppard, R.J, and Watson, R.J.

Notes: In this paper the authors report on the discovery, binding mode, and structure:activity relationship of the first potent, selective series of inhibitors of the BRPF1 (bromodomain and PHD finger-containing)  bromodomain.  Bromodomains are specific protein modules present in a group of chromatin-regulator proteins responsible for “reading” acetylated lysine residues. Although some bromodomain-containing proteins (BCPs), such as those in the BET subfamily, are well characterized and have been identified as potential therapeutic targets, other BCPs, including those in the BPRF subfamily, are less well understood.  These authors set out to generate selective inhibitors of the BRPF1 domain in order to better understand the functional role of this specific bromodomain region. Using an inhibitor discovery strategy based on other known compound-bromodomain interactions, a potent, selective inhibitor of the BRPF1 bromodomain was identified, synthesized, and characterized using in vitro methods.  To demonstrate the function of this compound in live cells, the NanoBRET™ assay for protein:protein interactions (PPI) was used. The NanoBRET™ PPI assay enabled the authors to demonstrate both the cell permeability of the newly identified compound and also the ability of the compound to disrupt chromatin binding of the BRPF1 domain. NanoLuc® Luciferase-tagged BRPF1 bromodomain and HaloTag®-labeled Histone H3.3 were used for the NanoBRET™ assay in HEK293 cells. Dose-response curves performed with the NanoBRET™ assay enabled calculation of the cellular IC50 of the newly identified compound. A less active control analog compound was unable to inhibit the BRPF1 bromodomain:Histone H3.3 interaction, demonstrating assay specificity. Finally, the newly identified compound was inactive in NanoBRET™ assays using a second BRPF1 isoform containing a natural insertion, a result that was consistent with the proposed compound mode of action. Confirmation that the new identified compound can enter cells and disrupt the BRPF1 bromodomain:chromatin interaction in a cellular environment suggests that it may be a useful compound for studying the physiological role and therapeutic potential of BCPs containing the BRPF1 bromodomain. (4514)

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Proc. Natl. Acad. Sci. USA 112, 148–153. Activation of Rab8 guanine nucleotide exchange factor Rabin8 by ERK1/2 in response to EGF signaling. 2014

Wang, J., Ren, J., Wu, B., Feng, S., Cai, G., Tuluc, F., Peränen, J. and Guo, W.

Notes: To investigate whether protein conformation of Rabin8 plays a role in autoinhibition, the authors created a Rabin8 fusion construct with NanoLuc® luciferase at the N terminus and HaloTag® protein at the C terminus so that they can use BRET as an indication of protein conformation. A t-SRARE protein, syntaxin-4 (STX4), which is known to have a closed conformation, was constructed with the same NanoLuc® luciferase-STX4-HaloTag® protein configuration for use as a positive control. Both the control STX4 protein and Rabin8 were expressed in E. coli, the NanoBRET™ Nano-Glo® Substrate added and fluorescence measured. As a negative control, TEV protease was used to cleave the HaloTag® sequence from the protein fusions, eliminating the NanoBRET™ signal. NanoBRET™ signals were determined from experiments comparing Rabin8 with a gain-of-function Rabin8 mutant, exposing Rabin8 to constitutively active ERK2 or a kinase-dead ERK2 and assessing wildtype Rabin8 versus Rabin8-4D, where the aspartates acted as phosphorylation mimics. (4566)

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Nucl. Acids Res. 42, e28. DNA transposition by protein transduction of the piggyBac transposase from lentiviral Gag precursors. 2014

Cai, Y., Bak, R.O., Krogh. L.B., Staunstrup, N.H., Moldt, B., Corydon, T.J., Schrøder, L.D. and Mikkelsen, J.G.

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)

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Appl. Environ. Microbiol. 80, 1150–1158. Ingestibility, digestibility, and engineered biological control potential of Flavobacterium hibernum, isolated from larval mosquito habitats. 2014

Chen, S., Kaufman, M.G., Korir, M.L. and Walker, E.D.

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)

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J. Virol. 88, 2034–2046. Stable, high-level expression of reporter proteins from improved alphavirus expression vectors to track replication and dissemination during encephalitic and arthritogenic disease. 2014

Sun, C., Gardner, C.L., Watson, A.M., Ryman, K.D. and Klimstra, W.B.

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)

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Proc. Natl. Acad. Sci. USA 111(38), 13990–5. Tyrosine phosphorylation of GluK2 up-regulates kainate receptor-mediated responses and downstream signaling after brain ischemia 2014

Zhu, Q.J., Kong, F.S., Xu, H., Wang, Y., Du, C.P., Sun, C.C., Liu, Y., Li, T. and Hou, X.Y.

Notes: In this study the authors looked for molecular mechanisms underlying the role of kainite receptors in ischemic stroke. In their studies, the researchers examined binding of Src kinase to GluK2, and the site of this interaction. A GST pulldown assay confirmed a direct interaction between GluK2 and Src in vitro. Then a bioluminescence resonance energy transfer (BRET) assay was used to examine this GluK2-Src interaction in live HEK293 cells, using NanoLuc® Luciferase as the energy donor, and HaloTag-labeled GluK2 as the energy acceptor. As reported, the co-expression of NLuc and HaloTag® fusions resulted in a significant NanoBRET ratio. The authors added untagged GluK2 as a competitor of the GluK2-HaloTag and Src-NLuc interaction, which resulted in reduction of the NanoBRET ratio. These results demonstrated that GluK2 interacts directly with Src in living cells. The GloMax® Discover Detection System was used to measure the output of these assays.

Their source of NanoLuc® Luciferase and HaloTag® tags was the NanoBRET™ PPI Starter System (Cat.# N1811, N1821). (4696)

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FEBS J. 280, 1773-81. A convenient luminescence assay of ferroportin internalization to study its interaction with hepcidin. 2013

Song, G., Jiang, Q., Xu, T., Liu, Y.L., Xu, Z.G., and Guo, Z.Y.

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)

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