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Biochem. Biophys. Rep. 12, 40–5. Application of a novel HiBiT peptide tag for monitoring ATF4 protein expression in Neuro2a cells. 2017

Oh-Hashi, K., Furuta, E., Fujimura, K. and Hirata, Y.

Notes: The authors used CRISPR/Cas9 editing to tag endogenous ATF4 with HiBiT and then studied changes in abundance following various cell treatments. (4928)

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J. Lipid Res. 58, 1722–1729. Cell-based, bioluminescent assay for monitoring the interaction between PCSK9 and the LDL receptor. 2017

Duellman, S.J., Machleidt, T., Cali, J.J. and Vidugiriene, J.

Notes: Researchers used NanoLuc® Luciferase technology to monitor the interaction between and LDLR and PCSK9. (4931)

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ACS Chemical Biology Epub before print. CRISPR-Mediated Tagging of Endogenous Proteins with a Luminescent Peptide 2017

Schwinn, M.K., Machleidt, T., Zimmerman, K., Eggers, C.T., Dixon, A.S., Hurst, R., Hall, M.P., Encell, L.P., Binkowski, B.F. and Wood, K.V.

Notes: Using CRISPR/Cas9 gene editing, HiBiT was tagged to the C terminus of HIF1α and several of its downstream transcriptional target proteins. The Nano-Glo® Lytic Detection System was used to quantify HiBiT-tagged endogenous protein levels, and the Nano-Glo® Live Cell Assay System was used for real-time detection in live cells. Bioluminescence was detected using the GloMax® Discover System. This study demonstrated the ability to efficiently tag endogenous proteins with HiBiT, allowing fast and sensitive quantification of the response dynamics in their regulated expression and covalent modifications. (4869)

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Anal. Biochem. 522, 10-17. Development of a bimolecular luminescence complementation assay for RGS: G protein interactions in cells. 2017

Bodle, C.R., Hayes, M.P., O'Brien, J.B. and Roman, D.L.

Notes: NanoBit™ technology was used to characterize interactions between regulators of G protein signaling proteins (RGS proteins) and target G proteins in this study. (4898)

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Virus Res. 243, 69–74. Development of a rapid and quantitative method for the analysis of viral entry and release using the NanoLuc® luciferase complementation assay. 2017

Sasaki, M., Anindita, P.D., Phongphaew, W., Carr, M., Kobayashi, S., Orba, Y. and Sawa, H.

Notes: The authors developed quantitative methods for the detection of cellular entry and release of subviral and flavivirus-like particles (SVPs, VLPs) by tagging the particles with HiBiT. The HiBiT tag was used for quantitation of the particles, and cellular entry was studied using a LgBiT-expressing stable Vero cell line. (4930)

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J. Immunol. Methods 450, 17-26. Development of NanoLuc® bridging immunoassay for detection of anti-drug antibodies 2017

Nath, N., Flemming, R., Godat, B., and Urh, M.

Notes: This article describes the development of a new bridging immunoassay where NanoLuc® luciferase enzyme is used as an antibody label. (4901)

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Cell Biochem. Funct. 34(7), 497-504.
SOD1 dimerization monitoring using a novel split NanoLuc, NanoBit.

Oh-Hashi K., Hirata Y., and Kiuchi K.

Notes: These authors used a NanoBiT® complementation assay to investigate interactions between proteins implicated in development of amyotrophic lateral sclerosis (ALS). Mutations in superoxide dismutase 1 (SOD1) and in TAR‐binding protein 43 kDa (TDP43) have been associated with ALS. The authors observed formation of SOD1 homodimers in neuronal cells transfected with SOD1 tagged with NanoBiT complementation partners, and found that SOD1 mutants associated with ALS were unable to form homodimers.  Next, possible interactions between SOD1 and TDP43 were assessed. When NanoBiT-tagged constructs of TBP43 and SOD were transfected into Neuro 2A cells, only NanoBiT-tagged SOD generated a luciferase signal. TBP43 did not self-associate to form homodimers, and SOD1 and TBP43 did not interact with each other to form heterodimers. The authors state that NanoBiT is a useful and sensitive tool for analyzing protein interactions under physiological conditions, and may provide a convenient way to monitor the modulation of SOD1 conformation by candidate treatments. (4765)

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Nat. Biotechnol. 34, 760-767. A bright cyan-excitable orange fluorescent protein facilitates dual-emission microscopy and enhances bioluminescence imaging in vivo. 2016

Chu, J., Oh, Y., Sens, A., Ataie, N., Dana, H., Macklin, J.J., Laviv, T., Welf, E.S., Dean, K.M., Zhang, F., Kim, B.B., Tang, C.T., Hu, M., Baird, M.A., Davidson, M.W., Kay, M.A., Fiolka, R., Yasuda, R., Kim, D.S., Ng, H.L. and Lin, M.Z.

Notes: The authors characterize a novel orange-red fluorescent protein (CyOFP1), which serves as an efficient acceptor for resonance energy transfer from NanoLuc® luciferase. An optimized fusion protein of CyOFP1 and NanoLuc® (Antares) was demonstrated to function as a highly sensitive bioluminescent reporter in vivo, producing substantially brighter signals from deep tissues than firefly luciferase and other bioluminescent proteins. During fluorometry, 50,000 cells in 10 μL Live Cell Imaging Solution were loaded into glass capillary tubes and gently pelleted by centrifugation for 15 s at 500g. 10 μL of Live Cell Imaging Solution with 20 μM D-luciferin, coelenterazine, ViviRen, or furimazine was then added. For BRET6 and Antares-expressing mice, tail vein injections were performed with 333 nmol (123 μg) of furimazine in 120 μL of 8% glycerol, 12% ethanol, 10% hydroxypropyl-β-cyclodextrin, and 35% polyethylene glycol 400 in water. (4760)

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Nucl. Acids Res. 44, 2348-2361. A short conserved motif in ALYREF directs cap- and EJC-dependent assembly of export complexes on spliced mRNAs 2016

Gromadzka, A.M., Steckelberg, A.-L., Singh, K.K, Hofmann, K. and Gehring, N.H.

Notes: NanoLuc® and Firefly luciferase reporters (pCMV128-NanoLuc and pCI-FireflyLuc) were co-transfected into HeLa cells to provide complementary measures of specific mRNA splicing and export activity. In the presence of splicing activity, firefly luciferase pre-mRNA is spliced in the nucleus, exported and translated resulting in firefly luciferase activity. In contrast, the open reading frame of the Nanoluc luciferase in pCMV128 NanoLuc® was placed between two weak splice sites. If the pre-mRNA is spliced, the NanoLuc coding sequence is removed and no luciferase can be expressed. The NanoLuc® reporter can only be translated when the unspliced pre-mRNA is transported to the cytoplasm. pCMV128 NanoLuc® was generated by inserting the NanoLuc® sequence into the NotI and BamHI sites of pCMV128, and then used for export assays. Cells were harvested 72 h post-transfection using 300 μl Passive Lysis Buffer. Lysates were analyzed using the NanoGlo® Dual-Luciferase® Reporter Assay. (4744)

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Malaria Journal 15, 232. An ultrasensitive NanoLuc-based luminescence system for monitoring Plasmodium berghei throughout its life cycle. 2016

De Niz, M., Stanway, R.R., Wacker, R., Keller, D. and Heussler, V.T.

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)

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Genes Cells 12401, [Epub ahead of print]. Application of NanoLuc to monitor the intrinsic promoter activity of GRP78 using the CRISPR/Cas9 system 2016

Oh-Hashi, K., Furuta, E., Norisada, J., Amaya, F., Hirata, Y. and Kiuchi, K.

Notes: CRISPR/Cas9 editing was used to tag the GRP78 gene with NanoLuc® luciferase in order to create a reporter of the intrinsic promoter activity of this gene. The authors used a single guide RNA against the N-terminal coding region of exon1 of the human GRP78 gene and constructed a donor gene that contains the puromycin-resistant gene following the NanoLuc® gene. The NanoLuc® gene was aligned with the puromycin-resistant gene through the 2A peptide sequence and was inserted into the pGL3-based vector (NL-2a-Puro-pGL3). To generate the donor gene, G78-NL, the N-terminal coding region that includes exon 1 of human GRP78 was inserted into the NanoLuc® gene in the NL-2a-Puro-pGL3. After lysis of the cells expressing the indicated gene in 24- or 96-well plates with passive lysis buffer, the lysate was mixed with the NanoLuc® substrate, and the luciferase activity in each well was measured using a GloMax® luminometer. In some cases, the culture medium from each well was mixed with the NanoLuc® substrate, and the activity for each was measured. (4754)

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PLos ONE 11, e0161930. Development of a Quantitative BRET Affinity Assay for Nucleic Acid-Protein Interactions 2016

Vickers, T.A. and Crooke, S.T.

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)

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Sci. Rep. 6, 28231. Fast and high resolution single-cell BRET imaging. 2016

Goyet, E., Bouquier, N., Ollendorff, V. and Perroy, J.

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)

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ACS Med. Chem. Lett. 7(5), 531–6. Fragment-based discovery of a selective and cell-active benzodiazepinone CBP/EP300 bromodomain inhibitor (CPI-637). 2016

Taylor, A.M., Côté, A., Hewitt, M.C., Pastor, R., Leblanc, Y., Nasveschuk, C.G., Romero, F.A., Crawford, T.D., Cantone, N., Jayaram, H., Setser, J., Murray, J., Beresini, M.H., de Leon Boenig, G., Chen, Z., Conery, A.R., Cummings, R.T., Dakin, L.A., Flynn, E.M., Huang, O.W., Kaufman, S., Keller, P.J., Kiefer, J.R., Lai, T., Li, Y., Liao, J., Liu, W., Lu, H., Pardo, E., Tsui, V., Wang, J., Wang, Y., Xu, Z., Yan, F., Yu, D., Zawadzke, L., Zhu, X., Zhu, X., Sims, R.J., 3rd, Cochran, A.G., Bellon, S., Audia, J.E., Magnuson, S. and Albrecht, B.K.

Notes: Researchers set out to identify CBP/EP300 bromodomain inhibitors potent to both in vitro targets and targets in cellular target engagement assays. They developed a series of selective probes of CBP/EP300 bromodomains identified first by fragment screening. They next substituted and modified parts of the fragments to improve potency and selectivity.

To determine whether improvements in CBP bromodomain inhibition that were observed in their biochemical assay would translate to a cellular context, they used a bioluminescence resonance energy transfer assay, NanoBRET, where a small CBP/EP300 bromodomain inhibitor disrupted the interaction between a HaloTag-labeled histone and bromodomain conjugated to NanoLuc® Luciferase.

NanoBRET™ CBP/Histone H3.3 Interaction Assay (Cat.# N1850) and FuGENE® HD Transfection Reagent (Cat.# E2311) were used in the cell-based assay. (4717)

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Oncotarget 7, 8172-8183. Identification of approved and investigational drugs that inhibit hypoxia-inducible factor-1 signaling 2016

Hsu, C.W., Huang, R., Khuc, T., Shou, D., Bullock, J., Grooby, S., Griffin, S., Zou, C., Little, A., Astley, H. and Xia, M.

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)

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Sci. Rep. 11, 29648. Mechanism for insulin-like peptide 5 distinguishing the homologous relaxin family peptide receptor 3 and 4 2016

Hu, M.J., Shao, X.X., Wang, J.H., Wei, D., Guo, Y.Q., Liu, Y.L., Xu, Z.G. and Guo, Z.Y.

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)

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ACS Chemical Biology 11, 400–408. NanoLuc Complementation Reporter Optimized for Accurate Measurement of Protein Interactions in Cells. 2016

Dixon, A.S., Schwinn, M.K., Hall, M.P., Zimmerman, K., Otto, P., Lubben, T.H., Butler, B.L., Binkowski, B.F., Machleidt, T., Kirkland, T.A., Wood, M.G., Eggers, C.T., Encell, L.P., Wood, K.V.

Notes: These authors designed a complementation reporter for detecting protein interactions under physiological conditions in mammalian cells. The engineered reporter (NanoBiT) is based on NanoLuc luciferase and is composed of two small subunits (18kDa and 1.3kDa), which associate weakly. The formation of a luminescent signal is dependent on the interaction characteristics of the proteins onto which the subunits are attached. The paper demonstrates utility of the NanoBiT assay for detecting protein interactions in live cells using  FRB/FKBP as an example, shows that response dynamics are rapid and reversible, and also illustrates use of the system to measure the pharmacology of kinase inhibitors that induce interaction between BRAF and CRAF. (4585)

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Bioconjug. Chem. 27, 1175–1187. NanoLuc: A small luciferase is brightening up the field of bioluminescence.  2016

England, C.G., Ehlerding, E.B. and Cai, W.

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

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PLos ONE 11, e0159263. Novel Bioluminescent Binding Assays for Ligand–Receptor Interaction Studies of the Fibroblast Growth Factor Family 2016

Song, G., Shao, X.X., Wu, Q.P., Xu, Z.G., Liu, Y.L. and Guo, Z.Y.

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)

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Nat. Comm. 7, 11624. Nrf2 suppresses macrophage inflammatory response by blocking proinflammatory cytokine transcription 2016

Kobayashi, E.H., Suzuki, T., Funayama, R., Nagashima, T., Hayashi, M., Sekine, H., Tanaka, N., Moriguchi, T., Motohashi, H., Nakayama, K. and Yamamoto, M.

Notes: A NanoLuc® luciferase reporter was used to further understand the mechanism of Nrf2 inhibition of proinflammatory cytokine gene induction. Approximately 1 kb upstream region of the IL6 gene TSS containing the WT ARE (GCTGAGTCA) or mutated ARE (AGATCTGAC) was conjugated to the translation start site of the NanoLuc® gene in the pNL2.2 vector. 293T cells (2.0 × 103 cells per well) were plated in 96-well plates 24 h before transfection. The NanoLuc® reporter vectors were co-transfected with pQC-FLAG-hNRF2T80R (constitutively active NRF2) plasmid33 and pcDNA3-p65 plasmid. After 48 hours of the transfection, the luminescence was quantified and normalized using NanoGlo® Dual-Luciferase® Reporter Assay. (4752)

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epub ahead of print June 23, 2016. 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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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. Comm. 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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