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Journal articles on the topic 'Luciferase reporter assays'

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Published: 4 June 2021

Last updated: 9 February 2022

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Kenda, Maša, Jan Vegelj, Barbara Herlah, Andrej Perdih, Přemysl Mladěnka, and Marija Sollner Dolenc. "Evaluation of Firefly and Renilla Luciferase Inhibition in Reporter-Gene Assays: A Case of Isoflavonoids." International Journal of Molecular Sciences 22, no. 13 (2021): 6927. http://dx.doi.org/10.3390/ijms22136927.

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Firefly luciferase is susceptible to inhibition and stabilization by compounds under investigation for biological activity and toxicity. This can lead to false-positive results in in vitro cell-based assays. However, firefly luciferase remains one of the most commonly used reporter genes. Here, we evaluated isoflavonoids for inhibition of firefly luciferase. These natural compounds are often studied using luciferase reporter-gene assays. We used a quantitative structure–activity relationship (QSAR) model to compare the results of in silico predictions with a newly developed in vitro assay that enables concomitant detection of inhibition of firefly and Renilla luciferases. The QSAR model predicted a moderate to high likelihood of firefly luciferase inhibition for all of the 11 isoflavonoids investigated, and the in vitro assays confirmed this for seven of them: daidzein, genistein, glycitein, prunetin, biochanin A, calycosin, and formononetin. In contrast, none of the 11 isoflavonoids inhibited Renilla luciferase. Molecular docking calculations indicated that isoflavonoids interact favorably with the D-luciferin binding pocket of firefly luciferase. These data demonstrate the importance of reporter-enzyme inhibition when studying the effects of such compounds and suggest that this in vitro assay can be used to exclude false-positives due to firefly or Renilla luciferase inhibition, and to thus define the most appropriate reporter gene.

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Lai, Chunfai, Xin Jiang, and Xianqiang Li. "Development of Luciferase Reporter-Based Cell Assays." ASSAY and Drug Development Technologies 4, no. 3 (2006): 307–15. http://dx.doi.org/10.1089/adt.2006.4.307.

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Paguio, Aileen, Pete Stecha, Keith V. Wood, and Frank Fan. "Improved Dual-Luciferase Reporter Assays for Nuclear Receptors." Current Chemical Genomics 4 (May 26, 2010): 43–49. http://dx.doi.org/10.2174/1875397301004010043.

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Roelant, Christiaan H., David A. Burns, and Winfried Scheirer. "Accelerating the Pace of Luciferase Reporter Gene Assays." BioTechniques 20, no. 5 (1996): 914–17. http://dx.doi.org/10.2144/96205pf01.

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Kolb, Alfred J., and Kenneth Neumann. "Luciferase Measurements in High Throughput Screening." Journal of Biomolecular Screening 1, no. 2 (1996): 85–88. http://dx.doi.org/10.1177/108705719600100207.

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Luminescence assays are becoming more popular in high throughput screening (HTS) laboratories with the luciferase reporter gene being the most common. As with other assays that are adapted to HTS, improvements have been made to the luciferase assay to make it better suited to the requirements of HTS. For the luciferase reporter gene, these improvements included stabilization of the enzyme, increasing the half-life of the luminescence signal to 5 h, and eliminating separation steps (centrifugation and aliquot transfer) after cell lysis. The improved assay, LucLite, is homogeneous and is measured directly in the cell culture media. In addition to reagent improvements, a temperature-controlled, multidetector microplate counter, TopCount, can quickly and accurately measure luminescence signals.

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Hancock, Michael K., Myleen N. Medina, Brendan M. Smith, and Anthony P. Orth. "Microplate Orbital Mixing Improves High-Throughput Cell-Based Reporter Assay Readouts." Journal of Biomolecular Screening 12, no. 1 (2006): 140–44. http://dx.doi.org/10.1177/1087057106296046.

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Reporter assays are commonly used for high-throughput cell-based screening of compounds, cDNAs, and siRNAs due to robust signal, ease of miniaturization, and simple detection and analysis. Among the most widely used reporter genes is the bioluminescent enzyme luciferase, which, when exposed to its substrate luciferin upon cell lysis, yields linear signal over a dynamic range of several orders of magnitude. Commercially available luciferase assay formulations have been developed permitting homogeneous, single-step cell lysis and reporter activity measurements. Assay conditions employed with these formulations are typically designed to minimize well-to-well luminescence variability due to variability in dispensing, evaporation, and incomplete sample mixing. The authors demonstrate that incorporating a microplate orbital mixing step into 96- and 384-well microplate cell-based luciferase reporter assays can greatly improve reporter readouts. They have found that orbital mixing using commercially available mixers facilitates maximal luciferase signal generation from high cell density–containing samples while minimizing variability due to partial cell lysis, thereby improving assay precision. The authors fully expect that widespread availability of mixers with sufficiently small orbits and higher speed settings will permit gains in signal and precision in the 1536-well format as well.

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Didiot, Marie-Cecile, Sergio Serafini, Martin J. Pfeifer, Frederick J. King, and Christian N. Parker. "Multiplexed Reporter Gene Assays: Monitoring the Cell Viability and the Compound Kinetics on Luciferase Activity." Journal of Biomolecular Screening 16, no. 7 (2011): 786–93. http://dx.doi.org/10.1177/1087057111407768.

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High-throughput screening assays with multiple readouts enable one to monitor multiple assay parameters. By capturing as much information about the underlying biology as possible, the detection of true actives can be improved. This report describes an extension to standard luciferase reporter gene assays that enables multiple parameters to be monitored from each sample. The report describes multiplexing luciferase assays with an orthogonal readout monitoring cell viability using reduction of resazurin. In addition, this technical note shows that by using the luciferin substrate in live cells, an assay time course can be recorded. This enables the identification of nonactive or unspecific compounds that act by inhibiting luciferase, as well as compounds altering gene expression or cell growth.

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Repele, Andrea, and Manu. "Robust Normalization of Luciferase Reporter Data." Methods and Protocols 2, no. 3 (2019): 62. http://dx.doi.org/10.3390/mps2030062.

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Transient Luciferase reporter assays are widely used in the study of gene regulation and intracellular cell signaling. In order to control for sample-to-sample variation in luminescence arising from variability in transfection efficiency and other sources, an internal control reporter is co-transfected with the experimental reporter. The luminescence of the experimental reporter is normalized against the control by taking the ratio of the two. Here we show that this method of normalization, “ratiometric”, performs poorly when the transfection efficiency is low and leads to biased estimates of relative activity. We propose an alternative methodology based on linear regression that is much better suited for the normalization of reporter data, especially when transfection efficiency is low. We compare the ratiometric method against three regression methods on both simulated and empirical data. Our results suggest that robust errors-in-variables (REIV) regression performs the best in normalizing Luciferase reporter data. We have made the R code for Luciferase data normalization using REIV available on GitHub.

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Siebring-van Olst, Ellen, Christie Vermeulen, Renee X. de Menezes, Michael Howell, Egbert F. Smit, and Victor W. van Beusechem. "Affordable Luciferase Reporter Assay for Cell-Based High-Throughput Screening." Journal of Biomolecular Screening 18, no. 4 (2012): 453–61. http://dx.doi.org/10.1177/1087057112465184.

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The firefly luciferase gene is commonly used in cell-based reporter assays. Convenient luciferase assay reagents for use in high-throughput screening (HTS) are commercially available. However, the high cost of these reagents is not within the means of some academic laboratories. Therefore, we set out to develop an affordable luciferase assay reagent applicable in an HTS format using simple liquid-handling steps. The reagent was homemade from individual chemical components and optimized for luminescence intensity and stability. We determined the minimal concentrations of the most expensive components, dithiothreitol (DTT) and D-luciferin, resulting in a total assay reagent cost of less than 1 cent per sample. Signal stability was maximized by omission of coenzyme A and reduction of DTT concentration. The assay was validated in a high-throughput setting using two cancer cell lines carrying a p53-dependent luciferase reporter construct and siRNAs modulating p53 transcriptional activity. Induction of p53 activity by silencing PPM1D or SYVN1 and reduction of p53 activity by silencing p53 remained constant over a 2-h measurement period, with good assay quality (Z′ factors mostly above 0.5). Hence, the luciferase assay described herein can be used for affordable reporter readout in cell-based HTS.

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Braeuning, Albert, та Silvia Vetter. "The nuclear factor κB inhibitor (E)-2-fluoro-4′-methoxystilbene inhibits firefly luciferase". Bioscience Reports 32, № 6 (2012): 531–37. http://dx.doi.org/10.1042/bsr20120043.

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Photinus pyralis (firefly) luciferase is widely used as a reporter system to monitor alterations in gene promoter and/or signalling pathway activities in vitro. The enzyme catalyses the formation of oxyluciferin from D-luciferin in an ATP-consuming reaction involving photon emission. The purpose of the present study was to characterize the luciferase-inhibiting potential of (E)-2-fluoro-4′-methoxystilbene, which is known as a potent inhibitor of the NF-κB (nuclear factor κB) signalling pathway that is used to modulate the NF-κB signalling pathway in vitro. Results show that (E)-2-fluoro-4′-methoxystilbene effectively inhibits firefly luciferase activity in cell lysates and living cells in a non-competitive manner with respect to the luciferase substrates D-luciferin and ATP. By contrast, the compound has no effect on Renilla and Gaussia luciferases. The mechanism of firefly luciferase inhibition by (E)-2-fluoro-4′-methoxystilbene, as well as its potency is comparable to its structure analogue resveratrol. The in vitro use of trans-stilbenes such as (E)-2-fluoro-4′-methoxystilbene or resveratrol compromises firefly luciferase reporter assays as well as ATP/luciferase-based cell viability assays.

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Unterreiner, Vincent, Yvonne Ibig-Rehm, Marjo Simonen, Hanspeter Gubler, and Daniela Gabriel. "Comparison of Variability and Sensitivity between Nuclear Translocation and Luciferase Reporter Gene Assays." Journal of Biomolecular Screening 14, no. 1 (2008): 59–65. http://dx.doi.org/10.1177/1087057108328016.

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High-content screening (HCS), a technology based on subcellular imaging by automated microscopy and sophisticated image analysis, has emerged as an important platform in small-molecule screening for early drug discovery. To validate a subcellular imaging assay for primary screening campaigns, an HCS assay was compared with a non—image-based readout in terms of variability and sensitivity. A study was performed monitoring the accumulation of the forkhead transcription factor of the O subfamily (FOXO3a) coupled with green fluorescent protein in the nucleus of human osteosarcoma (U-2 OS) cells. In addition, the transcription of a luciferase gene coupled with a FOXO3a-responsive promoter was monitored. This report demonstrates that both assay formats show good reproducibility in primary and concentration response screening despite differences in statistical assay quality. In primary screening, the correlation of compound activity between the 2 assays was low, in contrast to the good correlation of the IC50 values of confirmed compounds. Furthermore, the high-content imaging assay showed a mean shift of 2.63-fold in IC50 values compared with the reporter gene assay. No chemical scaffold was specifically found with 1 of the technologies only, however these results validate the HCS technology against established assays for screening of new molecular entities. ( Journal of Biomolecular Screening 2009:59-65)

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Bronstein, I., C. S. Martin, J. J. Fortin, C. E. Olesen, and J. C. Voyta. "Chemiluminescence: sensitive detection technology for reporter gene assays." Clinical Chemistry 42, no. 9 (1996): 1542–46. http://dx.doi.org/10.1093/clinchem/42.9.1542.

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Abstract A series of enzyme-activated chemiluminescence-based assays of reporter gene expression, useful in many biomedical applications, has been developed. The chemiluminescence detection systems for beta-galactosidase, beta-glucuronidase (GUS), and secreted placental alkaline phosphatase (SEAP) reporter enzymes are all based on use of 1,2-dioxetane substrates. This detection technology also permits the combined luminescence detection of two different reporter enzymes in the same tube, e.g., a dual assay for beta-galactosidase and luciferase. The sensitivity of these chemiluminescence assays is several orders of magnitude greater than that of conventional colorimetric or fluorometric detection methods; e.g., the detection limit for beta-galactosidase by the chemiluminescence assay is 8 fg and by a fluorometric assay is 2 pg. Furthermore, chemiluminescence enables detection of beta-galactosidase, GUS, and SEAP enzyme concentrations over a dynamic range of more than five to six orders in magnitude. These assays offer highly sensitive, quantitative, rapid, nonisotopic detection of reporter enzymes that are widely used in both mammalian cells and plant cells.

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Mudge, SR, WR Lewis-Henderson, and RG Birch. "Comparison of Vibrio and Firefly Luciferases as Reporter Gene Systems for Use in Bacteria and Plants." Functional Plant Biology 23, no. 1 (1996): 75. http://dx.doi.org/10.1071/pp9960075.

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Luciferase genes from Vibrio harveyi (luxAB) and firefly (luc) were introduced into E. coli, Agrobacteriurn, Arabidopsis and tobacco. Transformed bacteria and plants were quantitatively assayed for luciferase activity using a range of in vitro and in vivo assay conditions. Both lux and luc proved efficient reporter genes in bacteria, although it is important to be aware that the sensitive assays may detect expression due to readthrough from distant promoters. LUX activity was undetectable by liquid nitrogen-cooled CCD camera assays on intact tissues of plants which showed strong luxAB expression by in vitro assays. The decanal substrate for the lux assay was toxic to many plant tissues, and caused chemiluminescence in untransformed Arabidopsis leaves. These are serious limitations to application of the lux system for sensitive, non-toxic assays of reporter gene expression in plants. In contrast, LUC activity was readily detectable in intact tissues of all plants with luc expression detectable by luminometer assays on cell extracts. Image intensities of luc-expressing leaves were commonly two to four orders of magnitude above controls under the CCD camera. Provided adequate penetration of the substrate luciferin is obtained, luc is suitable for applications requiring sensitive, non-toxic assays of reporter gene expression in plants.

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Shifera, Amde Selassie, and John A. Hardin. "Factors modulating expression of Renilla luciferase from control plasmids used in luciferase reporter gene assays." Analytical Biochemistry 396, no. 2 (2010): 167–72. http://dx.doi.org/10.1016/j.ab.2009.09.043.

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Nieuwenhuijsen, Bart W., Youping Huang, Yuren Wang, Fernando Ramirez, Gary Kalgaonkar, and Kathleen H. Young. "A Dual Luciferase Multiplexed High-Throughput Screening Platform for Protein-Protein Interactions." Journal of Biomolecular Screening 8, no. 6 (2003): 676–84. http://dx.doi.org/10.1177/1087057103258287.

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To study the biology of regulators of G-protein signaling (RGS) proteins and to facilitate the identification of small molecule modulators of RGS proteins, the authors recently developed an advanced yeast 2-hybrid (YTH) assay format for GαZand RGS-Z1. Moreover, they describe the development of a multiplexed luciferase-based assay that has been successfully adapted to screen large numbers of small molecule modulators of protein-protein interactions. They generated and evaluated 2 different luciferase reporter gene systems for YTH interactions, a Gal4 responsive firefly luciferase reporter gene and a Gal4 responsive Renilla luciferase reporter gene. Both the firefly and Renilla luciferase reporter genes demonstrated a 40-to 50-fold increase in luminescence in strains expressing interacting YTH fusion proteins versus negative control strains. Because the firefly and Renilla luciferase proteins have different substrate specificity, the assays were multiplexed. The multiplexed luciferase-based YTH platform adds speed, sensitivity, simplicity, quantification, and efficiency to YTH high-throughput applications and therefore greatly facilitates the identification of small molecule modulators of protein-protein interactions as tools or potential leads for drug discovery efforts.

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Hannah, Rita R., Martha L. Jennens-Clough, and Keith V. Wood. "MipTec Proceedings: Transcriptional Assay Screens Using Renilla Luciferase as an Internal Control." JALA: Journal of the Association for Laboratory Automation 3, no. 4 (1998): 41–44. http://dx.doi.org/10.1177/221106829800300406.

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In cell biology research and pharmaceutical discovery, physiological responses of mammalian cells are commonly screened using transcriptional assays. Although firefly luciferase is widely used because of its rapid and simple assay, greater precision can be achieved using a second reporter as an internal control. Renilla luciferase serves as an efficient internal control because it can be measured as easily and rapidly using the same instrument. The Dual-Luciferase™ Reporter (DLR™) Assay developed at Promega measures both reporters sequentially within each sample, which eliminates the need to separate the test sample into aliquots for each assay. The expression of each reporter is independently quantitated using selective assay conditions based on their distinctive chemical characteristics. The firefly luciferase is initiated first by the addition of LAR II to the sample or cell lysate. Following measurement of the luminescent output, the firefly reaction is rapidly quenched and the Renilla reaction is simultaneously activated by addition of Stop & Glo™ Reagent, and the luminescence is measured a second time. The DLR™ Assay allows quantitation of both reporters within 4 seconds per well using a 96-well luminometer equipped with two reagent injectors. Multi-well plates may be processed even more rapidly using a CCD-based imaging system.

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Rawson, Jonathan M. O., Alice Duchon, Olga A. Nikolaitchik, Vinay K. Pathak, and Wei-Shau Hu. "Development of a Cell-Based Luciferase Complementation Assay for Identification of SARS-CoV-2 3CLpro Inhibitors." Viruses 13, no. 2 (2021): 173. http://dx.doi.org/10.3390/v13020173.

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The 3C-like protease (3CLpro) of SARS-CoV-2 is considered an excellent target for COVID-19 antiviral drug development because it is essential for viral replication and has a cleavage specificity distinct from human proteases. However, drug development for 3CLpro has been hindered by a lack of cell-based reporter assays that can be performed in a BSL-2 setting. Current efforts to identify 3CLpro inhibitors largely rely upon in vitro screening, which fails to account for cell permeability and cytotoxicity of compounds, or assays involving replication-competent virus, which must be performed in a BSL-3 facility. To address these limitations, we have developed a novel cell-based luciferase complementation reporter assay to identify inhibitors of SARS-CoV-2 3CLpro in a BSL-2 setting. The assay is based on a lentiviral vector that co-expresses 3CLpro and two luciferase fragments linked together by a 3CLpro cleavage site. 3CLpro-mediated cleavage results in a loss of complementation and low luciferase activity, whereas inhibition of 3CLpro results in 10-fold higher levels of luciferase activity. The luciferase reporter assay can easily distinguish true 3CLpro inhibition from cytotoxicity, a powerful feature that should reduce false positives during screening. Using the assay, we screened 32 small molecules for activity against SARS-CoV-2 3CLpro, including HIV protease inhibitors, HCV protease inhibitors, and various other compounds that have been reported to inhibit SARS-CoV-2 3CLpro. Of these, only five exhibited significant inhibition of 3CLpro in cells: GC376, boceprevir, Z-FA-FMK, calpain inhibitor XII, and GRL-0496. This assay should greatly facilitate efforts to identify more potent inhibitors of SARS-CoV-2 3CLpro.

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Ohmiya, Yoshihiro. "Simultaneous multicolor luciferase reporter assays for monitoring of multiple genes expressions." Combinatorial Chemistry & High Throughput Screening 18, no. 10 (2015): 937–45. http://dx.doi.org/10.2174/1386207318666150917095903.

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Theile, Dirk, Adriana Spalwisz, and Johanna Weiss. "Watch out for reporter gene assays with Renilla luciferase and paclitaxel." Analytical Biochemistry 437, no. 2 (2013): 109–10. http://dx.doi.org/10.1016/j.ab.2013.02.026.

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Sims, Robert J., Andrew S. Liss, and Paul D. Gottlieb. "Normalization of Luciferase Reporter Assays under Conditions that Alter Internal Controls." BioTechniques 34, no. 5 (2003): 938–40. http://dx.doi.org/10.2144/03345bm07.

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Tombácz, Kata, Duncan Mwangi, Dirk Werling, and Amanda J. Gibson. "Comparison of cellular assays for TLR activation and development of a species-specific reporter cell line for cattle." Innate Immunity 23, no. 4 (2017): 329–35. http://dx.doi.org/10.1177/1753425917695445.

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PRRs are sentinels of the innate immune system, with TLRs being the most important. Assays for TLR ligand interactions have been used to gain insights into their function and signaling pathways. As significant differences exist between species with regard to ligand recognition, it is necessary to adapt these tools for TLRs of other species. In the present work, we describe a species-specific cell-based assay adapted for the analysis of single PRRs. Human embryonic kidney 293T cells were stably transfected with the NF-κB-inducible reporter gene secreted embryonic alkaline phosphatase (SEAP) together with bovine TLR2. We compared the SEAP response with an existing luciferase NF-κB reporter assay for correlation with IL-8 production. A dose-dependent response was detected upon stimulation using both methods with good correlation to IL-8 secretion. Lower stimulant concentrations were detected by SEAP assay than IL-8 secretion. The luciferase assay produced high non-specific background for all ligand concentrations. Of all assays tested, we found the bovine-specific SEAP reporter assay to be the most convenient and delivered results in the shortest time. The developed reporter cell line would lend well to rapid, high-throughput TLR ligand screening for cattle.

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Omokoko, Tana A., Uli Luxemburger, Shaheer Bardissi, et al. "Luciferase mRNA Transfection of Antigen Presenting Cells Permits Sensitive Nonradioactive Measurement of Cellular and Humoral Cytotoxicity." Journal of Immunology Research 2016 (2016): 1–13. http://dx.doi.org/10.1155/2016/9540975.

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Immunotherapy is rapidly evolving as an effective treatment option for many cancers. With the emerging fields of cancer vaccines and adoptive cell transfer therapies, there is an increasing demand for high-throughputin vitrocytotoxicity assays that efficiently analyze immune effector functions. The gold standard51Cr-release assay is very accurate but has the major disadvantage of being radioactive. We reveal the development of a versatile and nonradioactive firefly luciferasein vitrotranscribed (IVT) RNA-based assay. Demonstrating high efficiency, consistency, and excellent target cell viability, our optimized luciferase IVT RNA is used to transfect dividing and nondividing primary antigen presenting cells. Together with the long-lasting expression and minimal background, the direct measurement of intracellular luciferase activity of living cells allows for the monitoring of killing kinetics and displays paramount sensitivity. The ability to cotransfect the IVT RNA of the luciferase reporter and the antigen of interest into the antigen presenting cells and its simple read-out procedure render the assay high-throughput in nature. Results generated were comparable to the51Cr release and further confirmed the assay’s ability to measure antibody-dependent cell-mediated cytotoxicity and complement-dependent cytotoxicity. The assay’s combined simplicity, practicality, and efficiency tailor it for the analysis of antigen-specific cellular and humoral effector functions during the development of novel immunotherapies.

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Ji, Changhua, Jun Zhang, Nick Cammack, and Surya Sankuratri. "Development of a Novel Dual CCR5-Dependent and CXCR4-Dependent Cell-Cell Fusion Assay System with Inducible gp160 Expression." Journal of Biomolecular Screening 11, no. 1 (2005): 65–74. http://dx.doi.org/10.1177/1087057105282959.

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In the current study, a novel coreceptor-specific cell-cell fusion (CCF) assay system is reported. The system possesses the following features: dual CCR5-dependent and CXCR4-dependent CCF assays, all stable cell lines, inducible expression of gp160 to minimize cytotoxicity, robust luciferase reporter, and 384-well format. These assays have been validated using various known HIV entry inhibitors targeting various stages of the HIV entry/fusion process, including fusion inhibitors, gp120 inhibitors, CCR5 antagonists, CCR5 antibodies, and CXCR4 antagonists. IC 50data generated from this assay system were well correlated to that from the antiviral assays. The effects of DMSOon this assay systemwere assessed, and a 2-to 3-fold increase in luciferase activitywas observed in the presence of 0.05% to2% DMSO. Although cell-cell fusion efficiencywas enhanced, no changes in drug response kinetics for entry inhibitors were found in the presence of 0.1% or 0.5% DMSO. This assay system has been successfully used for the identification and characterization of thousands of CCR5 inhibitors.

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George, Samantha E., Peter J. Bungay, and Louise H. Naylor. "Evaluation of a CRE-Directed Luciferase Reporter Gene Assay as an Alternative to Measuring cAMP Accumulation." Journal of Biomolecular Screening 2, no. 4 (1997): 235–40. http://dx.doi.org/10.1177/108705719700200408.

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A CHO reporter cell line expressing the firefly luciferase gene under the control of six cAMP response elements (CREs) was used to investigate the relationship between cAMP accumulation and cAMP dependent reporter gene expression and therefore, to assess the reporter gene system as an alternative functional assay. Timecourse experiments showed that cAMP accumulation preceded luciferase expression and that longer incubations (>2 h) were required to gain results with the reporter gene system. However, forskolin concentration dose-response studies revealed a 100-fold amplification of the signal measured by luciferase expression compared with direct cAMP accumulation, indicating that the reporter gene system afforded greater sensitivity. EC50 values determined for agonist activation of an inhibitory (5-HTlB-like) G-protein-coupled receptor (GPCR) were the same, and for a stimulatory GPCR (calcitonin Cla-like) were 10-fold lower, using the reporter gene system compared to cAMP accumulation assays, indicating the suitability of the reporter system for measuring the activity of receptors differentially coupled to the cAMP pathway. The phorbol ester, PMA, and the Ca2+ ionophore, A23187, were able to potentiate forskolin-stimulated luciferase expression but not cAMP accumulation, suggesting that the former could also be used to monitor cross-talk between different signal transduction pathways at the level of gene transcription.

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Harrison, Ewen M., O. J. Garden, James A. Ross, and Stephen J. Wigmore. "Firefly luciferase terminally degraded by mild heat exposure: Implications for reporter assays." Journal of Immunological Methods 310, no. 1-2 (2006): 182–85. http://dx.doi.org/10.1016/j.jim.2005.11.022.

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Liu, Xuemei, Jeffrey A. Kramer, Yi Hu, James M. Schmidt, Jianghong Jiang, and Alan G. E. Wilson. "Development of a High-Throughput Human HepG2 Dual Luciferase Assay for Detection of Metabolically Activated Hepatotoxicants and Genotoxicants." International Journal of Toxicology 28, no. 3 (2009): 162–76. http://dx.doi.org/10.1177/1091581809337166.

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Hepatic toxicity remains a major concern for drug failure; therefore, a thorough examination of chemically induced liver toxicity is essential for a robust safety evaluation. Current hypotheses suggest that the metabolic activation of a drug to a reactive intermediate is an important process. In this article, we describe a new high-throughput GADD45β reporter assay developed for assessing potential liver toxicity. Most importantly, this assay utilizes a human cell line and incorporates metabolic activation and thus provides significant advantage over other comparable assays used to determine hepatotoxicity. Our assay has low compound requirement and relies upon 2 reporter genes cotransfected into the HepG2 cells. The gene encoding Renilla luciferase is fused to the CMV promoter and provides a control for cell numbers. The firefly luciferase gene is fused to the GADD45β promoter and used to report an increase in DNA damage. A dual luciferase assay is performed by measuring the firefly and Renilla luciferase activities in the same sample. Results are expressed as the ratio of the 2 luciferase activities; increases over the control are interpreted as evidence of stress responses. This mammalian dual luciferase reporter has been characterized with, and without, metabolic activation using positive and negative control agents. Our data demonstrate that this assay provides for an assessment of potential toxic metabolites, is adaptable to a high-throughput platform, and yields data that accurately and reproducibly detect hepatotoxicants.

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Mašek, Tomáš, Václav Vopalenský, and Martin Pospíšek. "The Luc2 gene enhances reliability of bicistronic assays." Open Life Sciences 8, no. 5 (2013): 423–31. http://dx.doi.org/10.2478/s11535-013-0151-z.

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AbstractLuciferases are prominent reporters in molecular and cellular biology investigations including miRNA target studies and the determination of Internal Ribosome Entry Site (IRES) activities in bicistronic assays. A majority of the current bicistronic vectors contain a firefly luciferase reporter as the second cistron. One reason for this is the presence of cryptic transcription start sites inside the luciferase gene. We present here an experimental evaluation of the cryptic transcription within the latest version of the firefly luciferase gene, luc2. Using flow cytometric analysis, we observed a negligible amount of cryptic transcriptional activity that was only slightly above the background of untransfected cells. Nevertheless, quantitative reverse transcription PCR experiments revealed a six-to-nine-fold gradual increase of transcription along the coding region of the gene. The level of cryptic transcription from the coding region of the improved luc2 firefly luciferase gene is significantly lower when compared to the luc+ gene. In summary, the luc2 better fulfills the requirements of bicistronic assays than the previous luc+ version. The observed low cryptic transcription activity in luc2 could be limiting only in cases where weak IRESs are studied.

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Jiang, Yong, Zhe Sun, and Tong Cun Zhang. "Function Analysis of Luciferase Reporter Plasmid with AQP1 Gene Promoter." Advanced Materials Research 396-398 (November 2011): 1486–89. http://dx.doi.org/10.4028/www.scientific.net/amr.396-398.1486.

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The aquaporins (AQP) are a family of homologous water transporting proteins that are expressed in many epithelial, endothelial and other tissues. Myocardin is important for SMC differentiation, but its precise role in regulating the initiation of AQP1 transcription activity is less clear. Function analysis of AQP1 promoter luciferase reporter plasmid will provide the theory basis for researching the function of transcription activity. In this study, the rat and human myocardin promoter luciferase reporter construct were successfully constructed. Then to determine whether AQP1 transcription activity were regulated by myocardin, luciferase repoeter assays were performed in COS-7 cells. The results illustrated that myocardin significantly activated rat and human AQP1 promoter. AQP1 promoter transactivity was inhibited by △Q. The present study provided the first evidence that myocardin may have an influence on the expression of AQP1 and reveal a basis of the mechanism transcriptional regulation of AQP1.

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Snewin, Valerie A., Marie-Pierre Gares, Peadar ÓGaora, Zahra Hasan, Ivor N. Brown, and Douglas B. Young. "Assessment of Immunity to Mycobacterial Infection with Luciferase Reporter Constructs." Infection and Immunity 67, no. 9 (1999): 4586–93. http://dx.doi.org/10.1128/iai.67.9.4586-4593.1999.

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ABSTRACT Protective immunity to mycobacterial infection is incompletely understood but probably involves the coordinated interaction of multiple cell types and cytokines. With the aim of developing assays that might provide a surrogate measure of protective immunity, we have investigated the use of recombinant mycobacteria carrying luciferase reporter enzymes to assess the effectiveness of antimycobacterial immunity in model systems. Measurement of luminescence was shown to provide a rapid and simple alternative to the counting of CFU as a means of monitoring mycobacterial viability. We describe optimization of a luciferase reporter strain of Mycobacterium tuberculosis and demonstrate its application for the study of mycobacterial interactions with host cells in tissue culture and the rapid assessment of vaccine efficacy in a murine model.

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Zhang, Tong Cun, Yao Meng, Nan Wang, et al. "Construction and Functional Analysis of Luciferase Reporter Plasmid Containing NF-H Gene Promoter." Advanced Materials Research 915-916 (April 2014): 942–46. http://dx.doi.org/10.4028/www.scientific.net/amr.915-916.942.

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NF-H (a member of neurofilaments) is a protein widely expressed in all kinds of neurons after birth and can be used as one of the symbols of mature neurons. Constuction of NF-H promoter luciferase reporter plasmid will provide the theory basis for researching the effect of other transcription factors on regulating NF-H transcription. Here, human NF-H promoter luciferase reporter plasmid were successfully constructed. Then the effects of some key transcription factors were investigated by luciferase reporter assays in COS-7 cells. The results showed that retinoid X receptor α (RXRα) can enhance transcriptional activity of NF-H. Furthermore, ERK 1/2 (extracellular signal-regulated kinase 1/2) and STAT3 (signal transducer and activator of transcription 3) also show obvious impact in activating NF-H transcription.

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Auld, Douglas S., Janaki Narahari, Pei-i. Ho, et al. "Characterization and Use of TurboLuc Luciferase as a Reporter for High-Throughput Assays." Biochemistry 57, no. 31 (2018): 4700–4706. http://dx.doi.org/10.1021/acs.biochem.8b00290.

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Murray, Robert W., Earline P. Melchior, Jeanne C. Hagadorn, and Keith R. Marotti. "Staphylococcus aureus Cell Extract Transcription-Translation Assay: Firefly Luciferase Reporter System for Evaluating Protein Translation Inhibitors." Antimicrobial Agents and Chemotherapy 45, no. 6 (2001): 1900–1904. http://dx.doi.org/10.1128/aac.45.6.1900-1904.2001.

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ABSTRACT The promoter for the Staphylococcus aureus capsule polysaccharide synthesis gene (cap1A) was cloned in front of the firefly luciferase gene for use in a cell extractS. aureus transcription-translation system. The assay is rapid, reproducible, and sensitive and has a lower background level than the radiolabeled amino acid incorporation translation assays. We present data evaluating a transcription inhibitor and a number of protein translation inhibitors in this system.

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Larocque, Louise, Alex Bliu, Ranran Xu, et al. "Bioactivity Determination of Native and Variant Forms of Therapeutic Interferons." Journal of Biomedicine and Biotechnology 2011 (2011): 1–11. http://dx.doi.org/10.1155/2011/174615.

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The traditional antiviral assays for the determination of interferon potency are reported to have considerable variability between and within assays. Although several reporter gene assays based on interferon-inducible promoter activities have been reported, data from comprehensive validation studies are lacking and few studies have been conducted to analyze the variant forms of interferons, which could have undesirable clinical implications. Here, a reporter gene assay employing a HEK293 cell line stably transfected with luciferase gene under the control of interferon-stimulated response element promoter was developed and validated. The assay was found to be more sensitive, with a larger detection range than the antiviral assay. Several cytokines tested did not interfere with the test, suggesting the assay possesses a certain degree of selectivity. Moreover, the robustness of the assay was demonstrated by minimal variations in the results generated by different analysts and cell passage number (up to 52 passages). Finally, the method was employed to analyze several interferon variants (interferon-α2a) and we found that the aggregated form has completely lost its potency; while a modest loss of bioactivity in oxidized interferon was observed (approx. 23%), the deamidated form essentially retained its activity.

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Kumkhaek, Chutima, Daniel Shriner, Ayo P. Doumatey, Charles N. Rotimi та Griffin P. Rodgers. "An Intergenic SNP in the β–globin Gene Cluster Is Associated with Hyperuricemia and Influences Gene Transcription in Vitro". Blood 124, № 21 (2014): 1362. http://dx.doi.org/10.1182/blood.v124.21.1362.1362.

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Abstract Hemolysis as a suggested cause of hyperuricemia is based upon the fact that red blood cells contain uric acid. The development of hyperuricemia also generates from an increased synthesis of nucleic acids occurring as part of the erythropoietic response to hemolysis in hemoglobin disorders such as sickle cell anemia, α-thalassemia, and β-thalassemia. In addition, multiple genome-wide association studies (GWAS) have reported significant association between uric acid levels and specific genomic loci. However, the mechanism of hyperuricemia still remains controversial and it is also unknown whether African Americans have higher prevalence of hyperuricemia due to genetic vs environmental risk factors. Here, we used joint admixture mapping and association testing to identify genetic variants associated with serum uric acid levels in African American. Interestingly, we detected 6 SNPs (rs2855125, rs2855126, rs11036415, rs11036496, rs4320977, and rs4348933) in an intergenic region of the β–globin cluster on chromosome 11 are associated with high levels of serum uric acid in populations of African ancestry. Next, we explored the potential regulatory role of intergenic SNPs associated with hyperuricemia using luciferase reporter gene activity assays and electrophoretic mobility shift assays (EMSA). Each SNP-containing DNA fragment was amplified by PCR using human genomic DNA and inserted into a firefly luciferase reporter vector, pGL3-basic vector. 293T or K562 cells were co-transfected with these constructs and a Renilla luciferase vector to control for transfection efficiency. Expression of firefly luciferase driven by each SNP-containing DNA fragment was measured by a dual luciferase reporter assay and normalized by Renilla luciferase expression. SNPs rs2855126, rs11036496 and rs4348933 on chromosome 11 had significantly greater expression levels of firefly luciferase than pGL3-basic-transfected cells in both 293T and K562 cells. Of these, the SNP rs2855126 ancestral C allele (associated with higher serum uric acid levels) showed significantly higher luciferase activity than the derived G allele. Functionally, the luciferase activities from these constructs were determined to be very similar in both cell lines used. Alleles altering expression were further assessed for binding of nuclear extracted proteins by EMSA. We found specific gel shift bands for SNPs rs2855126, rs11036496, and rs4348933, suggesting these SNPs are situated in the binding site of potential transcription factors. These data provide new insights into the potential contribution of imbalanced β-globin gene expression to hyperuricemia. Disclosures No relevant conflicts of interest to declare.

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Zhang, Tong Cun, Yue Wang, Xing Hua Liao, Nan Wang, and Hao Zhou. "Construction and Functional Analysis of Luciferase Reporter Plasmid Containing PCNA Gene Promoter." Applied Mechanics and Materials 556-562 (May 2014): 257–60. http://dx.doi.org/10.4028/www.scientific.net/amm.556-562.257.

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PCNA (proliferating cell nuclear antigen) is a protein related to tumor development, which has been used extensively in breast cancer diagnosis and prognosis. PCNA has proven to be a useful marker to evaluate cell proliferation and prognosis when combined with other breast cancer markers. Construction of PCNA promoter luciferase reporter plasmid will provide the theory basis for researching the effect of other transcription factors on regulating PCNA transcription. In this study, a human PCNA promoter luciferase reporter construct was generated by PCR amplification of PCNA promoter. The PCR fragment was digested and cloned into pGL3 vector. The promoter sequence was verified by sequencing. The results showed that luciferase reporter plasmids of PCNA promoter were successfully constructed. Then the effects of some key transcription factors, which play important roles in breast cancer cell proliferation, were investigated by luciferase reporter assays in MCF-7 cells. The results showed that ERα can enhance transcriptional activity of PCNA. Furthermore, 17-β-estradiol (E2) also shows an obvious impact in activating PCNA transcription. Our data illuminated that E2 enhances ERα-induced proliferation potential of MCF-7 cells by stimulating the transcriptional activity of PCNA. Our research will provide a model to screen some novel factors in regulating proliferation marker transcription.

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Michaels, Samantha A., Han-Wei Shih, Bailin Zhang, et al. "Methionyl-tRNA synthetase inhibitor has potent in vivo activity in a novel Giardia lamblia luciferase murine infection model." Journal of Antimicrobial Chemotherapy 75, no. 5 (2020): 1218–27. http://dx.doi.org/10.1093/jac/dkz567.

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Abstract Background Methionyl-tRNA synthetase (MetRS) inhibitors are under investigation for the treatment of intestinal infections caused by Giardia lamblia. Objectives To properly analyse the therapeutic potential of the MetRS inhibitor 1717, experimental tools including a robust cell-based assay and a murine model of infection were developed based on novel strains of G. lamblia that employ luciferase reporter systems to quantify viable parasites. Methods Systematic screening of Giardia-specific promoters and luciferase variants led to the development of a strain expressing the click beetle green luciferase. Further modifying this strain to express NanoLuc created a dual reporter strain capable of quantifying parasites in both the trophozoite and cyst stages. These strains were used to develop a high-throughput cell assay and a mouse infection model. A library of MetRS inhibitors was screened in the cell assay and Compound-1717 was tested for efficacy in the mouse infection model. Results Cell viability in in vitro compound screens was quantified via bioluminescence readouts while infection loads in mice were monitored with non-invasive whole-animal imaging and faecal analysis. Compound-1717 was effective in clearing mice of Giardia infection in 3 days at varying doses, which was supported by data from enzymatic and phenotypic cell assays. Conclusions The new in vitro and in vivo assays based on luciferase expression by engineered G. lamblia strains are useful for the discovery and development of new therapeutics for giardiasis. MetRS inhibitors, as validated by Compound-1717, have promising anti-giardiasis properties that merit further study as alternative therapeutics.

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Kotarsky, Knut, Liselotte Antonsson, Christer Owman, and Björn Olde. "Optimized reporter gene assays based on a synthetic multifunctional promoter and a secreted luciferase." Analytical Biochemistry 316, no. 2 (2003): 208–15. http://dx.doi.org/10.1016/s0003-2697(03)00082-4.

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Arain, T. M., A. E. Resconi, D. C. Singh, and C. K. Stover. "Reporter gene technology to assess activity of antimycobacterial agents in macrophages." Antimicrobial Agents and Chemotherapy 40, no. 6 (1996): 1542–44. http://dx.doi.org/10.1128/aac.40.6.1542.

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Reporter strains of Mycobacterium tuberculosis and Mycobacterium bovis BCG endogenously expressing firefly luciferase were used in bioluminescence assays to evaluate the activities of isoniazid and rifampin against mycobacteria sequestered in human macrophages. This methodology allowed the efficacy of antibiotics against intracellular mycobacteria to be assessed without the labor-intensive procedures and protracted incubation requirements associated with conventional CFU determinations.

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Wang, Fang. "miR-384 targets metadherin gene to suppress growth, migration, and invasion of gastric cancer cells." Journal of International Medical Research 47, no. 2 (2019): 926–35. http://dx.doi.org/10.1177/0300060518817171.

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Objective MicroRNA-384 (miR-384) has been reported to function as a tumor suppressor in multiple cancers; however, its role in gastric cancer (GC) remains unclear. Methods We measured expression levels of miR-384 in GC cell lines and in a normal gastric cell line (GES-1). The association between miR-384 and the metadherin gene ( MTDH) was assessed by luciferase reporter assay and western blot. The effects of the miR-384/MTDH axis on GC cell behaviors were measured by CCK-8, wound-healing, and transwell invasion assays. Results miR-384 was significantly downregulated in GC cell lines compared with normal gastric cells. MTDH was identified as a direct target of miR-384 by bioinformatics analysis, luciferase assay, and western blot. Functional assays demonstrated that miR-384 inhibited GC cell proliferation, migration, and invasion through targeting MTDH. Conclusion These results reveal that miR-384 acts as a tumor suppressor in GC and suggest that the miR-384/MTDH axis may be a potential therapeutic target for GC.

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WARNECKE, Christina, Tobias WILLICH, Johannes HOLZMEISTER, Serge P. BOTTARI, Eckart FLECK, and Vera REGITZ-ZAGROSEK. "Efficient transcription of the human angiotensin II type 2 receptor gene requires intronic sequence elements." Biochemical Journal 340, no. 1 (1999): 17–24. http://dx.doi.org/10.1042/bj3400017.

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To investigate mechanisms of human angiotensin II type 2 receptor (hAT2) gene regulation we functionally characterized the promoter and downstream regions of the gene. 5ʹ-Terminal deletion mutants from -1417/+100 to -46/+100 elicited significant but low functional activity in luciferase reporter gene assays with PC12W cells. Inclusion into the promoter constructs of intron 1 and the transcribed region of the hAT2 gene up to the translation start enhanced luciferase activity 6.7±1.6-fold and 11.6±1.7-fold (means±S.E.M.) respectively, whereas fusion of the promoter to the spliced 5ʹ untranslated region of hAT2 cDNA did not, which indicated an enhancement caused by intronic sequence elements. Reverse transcriptase-mediated PCR confirmed that the chimaeric hAT2-luciferase mRNA was regularly spliced in PC12W cells. A Northern blot analysis of transfected cells showed levels of luciferase mRNA expression consistent with the respective enzyme activities. Mapping of intron 1 revealed that a 12 bp sequence in the centre of the intron was required for the increase in promoter activity, whereas the 5ʹ adjacent intronic region mediated a decrease in luciferase activity. Mutation of the 12 bp region led to altered protein binding and markedly decreased luciferase activity. Cloned into a promoterless luciferase vector, a 123 bp intron 1 fragment was able to direct reporter gene expression to the same activity as occurred in conjunction with the 5ʹ flanking region. These results indicate that sequence elements in intron 1 are necessary for efficient transcription of hAT2. In reporter gene assays, intron 1 might by itself function as a promoter and initiate transcription from an alternative start point.

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Comley, John C. W., Tony Reeves, and Phil Robinson. "A 1536 Colorimetric SPAP Reporter Assay: Comparison with 96- and 384-Well Formats." Journal of Biomolecular Screening 3, no. 3 (1998): 217–25. http://dx.doi.org/10.1177/108705719800300308.

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We report the successful miniaturization of a functional cell-based reporter gene assay. Utilizing interleukin-1beta (IL-1/β)-induced secreted placental alkaline phosphatase (SPAP)-catalyzed colorimetric readout, we reduced the assay volume to 10 μl using a Greiner 1536-well microplate. Our experiences of assay development, liquid handling (using a Hydra® 96; Robbins Scientific, Sunnyvale, CA), and detection (using the SpectraImage and SpectraFluor-Plus plate readers, Tecan Austria GmbH, Grodig, Austria) in 1536 wells are discussed. The effect of a set of 1,280 compounds in this SPAP reporter assay were compared between 96-, 384-, and 1536-well formats and were shown to be very similar. We conclude that cell-based reporter gene assays using SPAP-catalyzed color readouts are sensitive and highly reproducible in 1536-well plates and should be considered as a cost-effective alternative to luciferase reporters for miniaturized assay formats. Finally, we review the prospects for the implementation of routine HTS in 1536-well plates based around the instrumentation investigated.

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Rong, Juan, Ian Pass, Paul W. Diaz, et al. "Cell-Based High-Throughput Luciferase Reporter Gene Assays for Identifying and Profiling Chemical Modulators of Endoplasmic Reticulum Signaling Protein, IRE1." Journal of Biomolecular Screening 20, no. 10 (2015): 1232–45. http://dx.doi.org/10.1177/1087057115600414.

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Endoplasmic reticulum (ER) stress activates three distinct signal transducers on the ER membrane. Inositol-requiring protein 1 (IRE1), the most conserved signal transducer, plays a key role in ER stress-mediated signaling. During ER stress, IRE1 initiates two discrete signaling cascades: the “adaptive” signaling cascade mediated by the XBP1 pathway and the “alarm” signaling cascade mediated by stress-activated protein kinase pathways. Fine-tuning of the balance between the adaptive and alarm signals contributes significantly to cellular fate under ER stress. Thus, we propose that the design of high-throughput screening (HTS) assays to selectively monitor IRE1 mediated-signaling would be desirable for drug discovery. To this end, we report the generation of stable human neural cell lines and development of cell-based HTS luciferase (Luc) reporter gene assays for the identification of pathway-specific chemical modulators of IRE1. We implemented a cell-based Luc assay using a chimeric CHOP-Gal4 transcription factor in 384-well format for monitoring IRE1 kinase-mediated p38MAPK activation and an unfolded response pathway element (URPE)–Luc cell-based assay in 1536-well format for monitoring IRE1’s RNase-mediated activation of XBP1. Chemical library screening was successfully conducted with both the CHOP/Gal4-Luc cells and UPRE-Luc engineered cells. The studies demonstrate the feasibility of using these HTS assays for discovery of pathway-selective modulators of IRE1.

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Ge, Yiman, Jia Shu, Gang Shi, Fuguo Yan, Yejing Li, and Hangliang Ding. "miR-100 Suppresses the Proliferation, Invasion, and Migration of Hepatocellular Carcinoma Cells via Targeting CXCR7." Journal of Immunology Research 2021 (July 17, 2021): 1–11. http://dx.doi.org/10.1155/2021/9920786.

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This study is to elucidate the functions of miR-100 in hepatocellular carcinoma progression and to explore the underlying mechanisms. Expression levels of miR-100 in normal-cancer hepatocellular carcinoma tissues were measured using quantitative real-time PCR (qRT-PCR). The invasive and proliferative abilities of hepatocellular carcinoma cell lines transfected with mimic-NC or mimic-miR-100 were measured using transwell, CCK-8, and colony formation assays. The binding sites between CXCR7 and miR-100 were determined using luciferase reporter assays. The correlation of CXCR7 and miR-100 in hepatocellular carcinoma progression was further confirmed by cotransfection assays. Our results showed that miR-100 was significantly lower expressed in hepatocellular carcinoma tissues and negatively associated with CXCR7 expression. Cell functional assays’ results found that upregulation of miR-100 inhibited the proliferative, invasive, and migrative abilities in hepatocellular carcinoma cells. Luciferase reporter assay suggested that CXCR7 mRNA and miR-100 bound one another. Increasing CXCR7 expression reversed the inhibitive effects of upregulated miR-100 in hepatocellular carcinoma cells. Further study showed that miR-100/CXCR7 played a role in hepatocellular carcinoma progression by regulating metalloproteinase-2 (MMP2) and vascular endothelial growth factor (VEGF). Conclusively, miR-100 exerts antitumor effects on hepatocellular carcinoma. Overexpression of miR-100 attenuates the invasive and proliferative abilities of hepatocellular carcinoma cells by targeting CXCR7.

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Maffia, Anthony M., Ilona Kariv, and Kevin R. Oldenburg. "Miniaturization of a Mammalian Cell-Based Assay: Luciferase Reporter Gene Readout in a 3 Microliter 1536-Well Plate." Journal of Biomolecular Screening 4, no. 3 (1999): 137–42. http://dx.doi.org/10.1177/108705719900400307.

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The combined efforts of the fields of combinatorial chemistry and genomics have significantly increased the number of compounds and therapeutic targets available for screening. The number of compounds will reach into the million range in the near future and provide vast chemical diversity for drug discovery. However, this reservoir of chemical diversity creates downstream hurdles for any screening effort. Properly examining this number of compounds increases investments dramatically, both in the number of dollars spent and amount of limited reagents depleted. Traditional HTS techniques, such as the use of 96-well microtiter plates, have paved the way for faster processing speeds, but are being rapidly overwhelmed by screening demands. Miniaturization of such assays will allow for greater throughput, while concurrently reducing cost. To date, miniaturization efforts have been most successfully applied to bacterial and soluble protein based assays. Questions about the ability to deliver microquantities of mammalian cells without disruption of the cell membrane and/or activation of stress responses have been raised. An assay has been developed in which a human T-cell screen has been adapted to a 1536-well plate format. Through the use of a luciferase reporter gene system, it is shown that a mammalian cell-based assay may be successfully performed in 3 pil and potent inhibitors of the target of interest identified.

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Becker, Jonas Philipp, Jannick Robert Clemens, Dirk Theile, and Johanna Weiss. "Bortezomib and ixazomib protect firefly luciferase from degradation and can flaw respective reporter gene assays." Analytical Biochemistry 509 (September 2016): 124–29. http://dx.doi.org/10.1016/j.ab.2016.06.015.

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Madeira, João L. O., Alexander A. L. Jorge, Regina M. Martin, et al. "A homozygous point mutation in the GH1 promoter (c.-223C>T) leads to reduced GH1 expression in siblings with isolated GH deficiency (IGHD)." European Journal of Endocrinology 175, no. 2 (2016): K7—K15. http://dx.doi.org/10.1530/eje-15-0149.

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Context Mutations in the GH1 promoter are a rare cause of isolated growth hormone deficiency (IGHD). Objective To identify the molecular aetiology of a family with IGHD. Design DNA sequencing, electromobility shift (EMSA) and luciferase reporter assays. Setting University Hospital. Patients Three siblings (2M) born to consanguineous parents presented with IGHD with normal pituitary on MRI. Methods The GH1 proximal promoter, locus control region, five exons and four introns as well as GHRHR gene were sequenced in genomic DNA by Sanger method. DNA–protein interaction was evaluated by EMSA in nuclear extracts of GH3 pituitary cells. Dual-luciferase reporter assays were performed in cells transiently transfected with plasmids containing four different combinations of GH1 allelic variants (AV). Results The patients harboured two homozygous variants (c.-185T>C and c.-223C>T) in the GH1 promoter within a highly conserved region and predicted binding sites for POU1F1/SP1 and SP1 respectively. The parents and brother were carriers and these variants were absent in 100 controls. EMSA demonstrated absent binding of GH3 nuclear extract to the c.-223C>T variant and normal binding of both POU1F1 protein and GH3 nuclear extract to the c.-185T>C variant. In contrast to GH1 promoter with AV only at c.-185, the GH1 promoter containing the AV only at c.-223 and at both positions drove significantly less expression of luciferase compared with the promoter containing either positions wild type in luciferase reporter assays. Conclusion To our knowledge, c.-223C>T is the first homozygous point mutation in the GH1 promoter that leads to short stature due to IGHD.

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Woo, Seung Kyoon, Min Seong Kwon, Zhihua Geng, et al. "Sequential Activation of Hypoxia-Inducible Factor 1 and Specificity Protein 1 is Required for Hypoxia-Induced Transcriptional Stimulation of Abcc8." Journal of Cerebral Blood Flow & Metabolism 32, no. 3 (2011): 525–36. http://dx.doi.org/10.1038/jcbfm.2011.159.

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Cerebral ischemia causes increased transcription of sulfonylurea receptor 1 (SUR1), which forms SUR1-regulated NC(Ca-ATP) channels linked to cerebral edema. We tested the hypothesis that hypoxia is an initial signal that stimulates transcription of Abcc8, the gene encoding SUR1, via activation of hypoxia-inducible factor 1 (HIF1). In the brain microvascular endothelial cells, hypoxia increased SUR1 abundance and expression of functional SUR1-regulated NC(Ca-ATP) channels. Luciferase reporter activity driven by the Abcc8 promoter was increased by hypoxia and by coexpression of HIF1α. Surprisingly, a series of luciferase reporter assays studying the Abcc8 promoter revealed that binding sites for specificity protein 1 (Sp1), but not for HIF, were required for stimulation of Abcc8 transcription by HIF1α. Luciferase reporter assays studying Sp1 promoters of three species, and chromatin immunoprecipitation analysis in rats after cerebral ischemia, indicated that HIF binds to HIF-binding sites on the Sp1 promoter to stimulate transcription of the Sp1 gene. We conclude that sequential activation of two transcription factors, HIF and Sp1, is required to stimulate transcription of Abcc8 following cerebral ischemia. Sequential gene activation in cerebral ischemia provides a plausible molecular explanation for the prolonged treatment window observed for inhibition of the end-target gene product, SUR1, by glibenclamide.

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Pathe-Neuschäfer-Rube, Andrea, Frank Neuschäfer-Rube, Gerald Haas, Nina Langoth-Fehringer, and Gerhard Püschel. "Cell-Based Reporter Release Assay to Determine the Potency of Proteolytic Bacterial Neurotoxins." Toxins 10, no. 9 (2018): 360. http://dx.doi.org/10.3390/toxins10090360.

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Despite the implementation of cell-based replacement methods, the mouse lethality assay is still frequently used to determine the activity of botulinum toxin (BoNT) for medical use. One explanation is that due to the use of neoepitope-specific antibodies to detect the cleaved BoNT substrate, the currently devised assays can detect only one specific serotype of the toxin. Recently, we developed a cell-based functional assay, in which BoNT activity is determined by inhibiting the release of a reporter enzyme that is liberated concomitantly with the neurotransmitter from neurosecretory vesicles. In theory, this assay should be suitable to detect the activity of any BoNT serotype. Consistent with this assumption, the current study shows that the stimulus-dependent release of a luciferase from a differentiated human neuroblastoma-based reporter cell line (SIMA-hPOMC1-26-GLuc cells) was inhibited by BoNT-A and-C. Furthermore, this was also inhibited by BoNT-B and tetanus toxin to a lesser extent and at higher concentrations. In order to provide support for the suitability of this technique in practical applications, a dose–response curve obtained with a pharmaceutical preparation of BoNT-A closely mirrored the activity determined in the mouse lethality assay. In summary, the newly established cell-based assay may represent a versatile and specific alternative to the mouse lethality assay and other currently established cell-based assays.

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Yin, Wei, Lei Shi, and Yanjiao Mao. "MicroRNA-449b-5p suppresses cell proliferation, migration and invasion by targeting TPD52 in nasopharyngeal carcinoma." Journal of Biochemistry 166, no. 5 (2019): 433–40. http://dx.doi.org/10.1093/jb/mvz057.

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Abstract Nasopharyngeal carcinoma (NPC) is an important type of head and neck malignant cancer with geographical distribution. MicroRNA-449b-5p (miR-449b-5p) is related to the development of various cancers, while its function in NPC remains unknown. The present study aimed to investigate the role and target gene of miR-449b-5p in NPC. Expressions of miR-449b-5p in NPC cell lines and clinical tissues were detected by quantitative real-time polymerase chain reaction (qRT-PCR). Cell proliferation was determined by MTT and colony formation assays. Migration and invasion abilities after different treatment were evaluated by wound healing and Transwell assays, respectively. Dual-luciferase reporter assay was performed to explore the relationship between miR-449b-5p and tumour protein D52 (TPD52). TPD52 expression was determined by qRT-PCR and western blot assay. miR-449b-5p was significantly downregulated in NPC cell lines and clinical tissues than the matched control. Overexpression of miR-449b-5p inhibited proliferation, migration and invasion of NPC cells. Dual-luciferase reporter assay indicated that miR-449b-5p directly targeted TPD52. Furthermore, shRNA-mediated downregulation of TPD52 rectified the promotion of cell migration and invasion by miR-449b-5p inhibition. In conclusion, the present study suggests that miR-449b-5p, as a novel tumour-suppressive miRNA against NPC, inhibits proliferation, migration and invasion of NPC cells via inhibiting TPD52 expression.

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Kemp, Daniel M., Samantha E. George, Toby C. Kent, Peter J. Bungay, and Louise H. Naylor. "The Effect of ICER on Screening Methods Involving CRE-Mediated Reporter Gene Expression." Journal of Biomolecular Screening 7, no. 2 (2002): 141–48. http://dx.doi.org/10.1177/108705710200700207.

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We describe a mechanism whereby increasing levels of cAMP in Chinese hamster ovary (CHO) and other cell lines lead to a significant repression in cAMP response element (CRE)-mediated luciferase reporter gene expression. This effect was shown to be mediated by a modulatory factor located downstream of cyclic AMP (cAMP), which displayed the temporal regulation pattern of an immediate early gene. The expression of this inducible cAMP early repressor (ICER) was shown to be coincident with the time and concentration dependency of the repression of CRE-mediated luciferase gene expression on the treatment of CHO cells with forskolin. Furthermore, this phenomenon was also observed in JEG and GH3 cell lines (both previously reported to express ICER), but not in COS-7 cells, which do not express ICER. These studies suggest that, in certain cell lines, expression of ICER can be induced at pharmacologically elevated cAMP levels, leading to a potent inhibition of CRE-mediated gene expression. We therefore conclude that screening methodologies employing such CRE-linked reporter genes (particularly in high-throughput screening assays) may produce false functional responses in certain cell lines. Moreover, such effects are likely to be exacerbated in screening assays in which receptors either are overexpressed or high concentrations of potent cAMP-elevating compounds are used.

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