Relevant bibliographies by topics / Reporter luciferase assay

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  1. Journal articles
  2. Dissertations / Theses
  3. Book chapters
  4. Conference papers

Academic literature on the topic 'Reporter luciferase assay'

Author: Grafiati
Published: 4 June 2021
Last updated: 11 February 2022

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Journal articles on the topic "Reporter luciferase assay"

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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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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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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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Wu, Chun, Chie Suzuki-Ogoh, and Yoshihiro Ohmiya. "Dual-reporter assay using two secreted luciferase genes." BioTechniques 42, no. 3 (2007): 290–92. http://dx.doi.org/10.2144/000112428.

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Cheng, Zhijie, Denise Garvin, Aileen Paguio, Pete Stecha, Keith Wood, and Frank Fan. "Luciferase Reporter Assay System for Deciphering GPCR Pathways." Current Chemical Genomics 4 (December 21, 2010): 84–91. http://dx.doi.org/10.2174/1875397301004010084.

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Barriscale, Kathy A., Sharon A. O’Sullivan, and Tommie V. McCarthy. "A single secreted luciferase-based gene reporter assay." Analytical Biochemistry 453 (May 2014): 44–49. http://dx.doi.org/10.1016/j.ab.2014.02.019.

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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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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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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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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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Dissertations / Theses on the topic "Reporter luciferase assay"

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Wiencierz, Anne Maria. "Entwicklung eines Dual-Luciferase-Reportergen-Assays zum Nachweis der Induktion antioxidativer Enzyme durch Nahrungsbestandteile." Master's thesis, Universität Potsdam, 2008. http://opus.kobv.de/ubp/volltexte/2009/2790/.

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Die Induktion antioxidativer Enzyme gilt als eine Möglichkeit, die antioxidative Kapazität von Zellen zu steigern und dadurch mit oxidativem Stress assoziierten Erkrankungen (z. B. Herz-Kreislauf-Erkrankungen, Neurodegeneration, Atherosklerose) vorzubeugen. Ausgehend davon wurde in der vorliegenden Arbeit der Dual-Luciferase-Reportergen-(DLR)-Assay zum Nachweis der Induktion der antioxidativen Enzyme Katalase (CAT), zytosolische Glutathion-Peroxidase (GPX1) und Kupfer-Zink-Superoxid-Dismutase (SOD1) entwickelt. Im Zuge dessen wurden drei Säugetierzelllinien (CaCo2, IEC-18, V79) auf ihre Eignung zur Modellzelllinie untersucht. Aufgrund der Transfektionseffizienz wurde die Fibroblastenzelllinie V79 ausgewählt. Zur Gewährleistung eines hohen Substanzdurchsatzes des DLR-Assays wurden bei der Etablierung Parameter wie Kulturplattenformat, DNA-Menge, Luciferasen-Kinetik berücksichtigt. Nach erfolgreicher Etablierung des Versuchs im 96-Well-Format wurden L-Carnitin, Catechin, Epigallocatechingallat, Genistein, Wasserstoffperoxid (H2O2), Natrium-Ascorbat, Paraquat, Quercetin, 12-O-Tetradecanoylphorbol-13-Acetat (TPA) und Trolox in nicht-zytotoxischen Konzentrationen hinsichtlich der Aktivierung des Ratten-CAT-, des humanen GPX1- und des humanen SOD1-Promotors untersucht. Die Bestimmung der maximal tolerierbaren Behandlungskonzentration erfolgte im Vorfeld mittels Resazurintest. Von den zehn Verbindungen zeichneten sich drei Substanzen als potente Induktoren für die SOD1 und die GPX1 aus. Die 24-stündige Behandlung von mit Reportergenkonstrukten transient transfizierten V79-Zellen mit 100 µM Paraquat resultierte in einer Verdopplung der relativen SOD1-Promotor-Aktivität und einer Erhöhung der relativen GPX1-Promotor-Aktivität auf 1,6 bzw. 1,7. Die Stimulation mit 20 µM Genistein oder 10 µM Quercetin führte wiederum zu einer Verdopplung bis Verdreifachung der relativen SOD1- und GPX1-Promotor-Aktivität. Der Promotor der Rattenkatalase konnte demgegenüber nur durch 50 µM H2O2 aktiviert werden (1,5fach). Für diesen DLR-Assays bieten sich folglich Genistein, Quercetin wie auch H2O2 als Referenzsubstanzen an. Um aber eine qualitative Charakterisierung der einzelnen Verbindungen hinsichtlich ihres Induktionspotentials zu gewährleisten, sollten von allen getesteten Substanzen Dosis-Wirkungskurven aufgenommen werden. Zudem wird für den routinemäßigen Einsatz die Verwendung stabil transfizierter Zellen zur Vermeidung von mit der Transfektion verbundenen experimentellen Schwankungen empfohlen.<br>The induction of antioxidative enzymes might be an opportunity to elevate the cellular antioxidative capacity and, thus, to prevent oxidative stress associated diseases (e. g. cardio-vascular disease, neurodegenerative disease, atherosclerosis). Based on this idea the dual luciferase reporter gene (DLR) assay was developed to demonstrate the induction of three antioxidative enzymes: catalase (CAT), cytosolic glutathione peroxidase (GPX1), and copper-zinc superoxide dismutase (SOD1). In the course of the development three mammalian cell lines (CaCo2, IEC-18, V79) were tested for their ability to serve as a model cell line. The line V79 was chosen due to the transfection efficiency. To give consideration to a high-throughput several parameters were studied (e. g. format of the cultural plates, amount of DNA, kinetics of the luciferases) and the DLR assay was successfully established in 96 well plates. Subsequently, L-carnitine, catechin, epigallocatechin gallate, genistein, hydrogen peroxide (H2O2), sodium ascorbate, paraquat, quercetin, 12-O-tetradecanoylphorbol-13-acetate (TPA) and trolox were tested in non-cytotoxic concentrations for the activation of the rat CAT, human GPX1 and human SOD1 promoter. The maximally tolerable concentrations were determined by resazurin test in advance. Three out of these ten compounds were identified as potent inducers of GPX1 and SOD1. Stimulation of reporter gene construct transient transfected V79 cells for 24 hours with 100 µM paraquat caused a duplication of the relative GPX1 promoter activity and a 1.6-/1.7-fold increase of the relative SOD1 promoter activity. The incubation with 20 µM gen-istein or 10 µM quercetin resulted in duplication to triplication of both, the relative GPX1 and SOD1 promoter activity. In contrast, the rat CAT promoter was activated by 50 µM H2O2 (1.5-fold). Consequently, genistein, quercetin, and H2O2 are considered to be suitable reference substances for this DLR assay. To further characterize the inducing potential of the tested compounds all of them should be tested in different concentrations. Furthermore, for the routinely performed DLR assay it is recommended to use stably transfected cells to eliminate transfection caused variations.
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Zheng, Qiaoyun. "Study of Cancer Related Proteins: LRG-1 and PD-L1." Cleveland State University / OhioLINK, 2017. http://rave.ohiolink.edu/etdc/view?acc_num=csu1495735827467521.

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Lai, John. "Functional analyses of polymorphisms in the promoters of the KLK3 and KLK4 genes in prostate cancer." Queensland University of Technology, 2006. http://eprints.qut.edu.au/16454/.

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This PhD aimed to elucidate the mechanisms by which polymorphisms may alter androgen-induced transactivation of androgen receptor (AR) target genes which may be important in prostate cancer aetiology. The second aspect of this PhD focused on identifying and characterising functional polymorphisms that may have utility as predictive risk indicators for prostate cancer and which may aid in earlier therapeutic intervention and better disease management. Analyses were carried out on the kallikrein-related peptidase 3 (KLK3), also known as the prostate specific antigen (PSA), gene and the kallikrein-related peptidase 4 (KLK4) gene. The PSA and KLK4 genes are part of the serine protease family that have trypsin or chymotrypsin like activity and are thought to play a role in the development of hormone-dependent cancers in tissues such as those in the prostate, breast, endometrium and ovaries. In the prostate, PSA is regulated by androgens and three androgen response elements (AREs) have been described in the promoter and upstream enhancer region. The PSA ARE I harbours a polymorphism at -158 bp from the transcription initiation site (TIS) that results in a G to A transition (G-158A). This PhD investigated the functional significance of the PSA G-158A polymorphism which has been reported to be associated with prostate cancer risk. Electromobility shift assays (EMSAs) investigating the interaction of ARE I variants with the AR DNA binding domain (AR-DBD) demonstrated that the A allele had a two-fold increased binding affinity for the AR-DBD when compared with the G allele. This was confirmed with endogenous AR in limited proteolysis-EMSA experiments. The limited proteolysis-EMSA experiments also demonstrated differential sensitivities of PSA ARE I alleles to trypsin digestion, which suggests that the G-158A polymorphism has an allosteric effect on the AR that alters AR/ARE I complex stability. Furthermore, Chromatin Immunoprecipitation (ChIP) assays suggest that the A allele more readily recruited the AR in vivo when compared with the G allele and is consistent with the in vitro binding data. Luciferase reporter assays carried out in both LNCaP and 22Rv1 prostate cancer cells, and using the natural (dihydrotestosterone; DHT) ligand demonstrated that the A allele was more responsive to androgens in LNCaP cells. Hence, this study has elucidated the potential mechanisms by which the G-158A polymorphism may differentially regulate PSA expression (of which up-regulation of PSA is thought to be important in prostate cancer development and progression). KLK4 has similar tissue-restricted expression as PSA and is up-regulated by steroid hormones in many endocrine cells including those in the prostate. A putative ARE (KLK4-pARE) located at -1,005 to -1019 relative to the more predominantly used transcription initiation site, TIS3, was initially found in supershift assays using AR antibodies to interact with endogenous AR. However, subsequent EMSA analysis using purified AR-DBD suggest that KLK4-pARE may be interacting with the AR indirectly. To investigate this hypothesis, a tandem construct of KLK4-pARE was cloned into the pGL3-Promoter vector for hormone-induced reporter assays. However, reporter assays did not demonstrate any responsiveness of KLK4-pARE to androgens, estradiol or progestins. Consequently, Real-Time PCR was carried out to reassess the hormonal regulation of KLK4 at the mRNA level. Consistent with the literature, data from this study suggests that KLK4 may be up-regulated by androgens, progestins and estradiol in a cyclical manner. Hormone-induced luciferase reporter assays were then carried out on seven promoter constructs that span 2.8 kb of the KLK4 promoter from TIS3. However, none of the seven promoter constructs demonstrated any significant responsiveness to androgens, estradiol or progestins. This study suggests that hormone response elements (HREs) that may drive the hormonal regulation of KLK4 in prostate cancer may be located further upstream from the promoter region investigated in this PhD, or alternatively, may lie 3' of TIS3. The characterisation of KLK4 promoter polymorphisms and their flanking sequences were also carried out in parallel to the functional work with the intent to assess the functional significance of any polymorphisms that may be located within HREs. In total 19 polymorphisms were identified from the public databases and from direct sequencing within 2.8 kb of the KLK4 promoter from TIS3. However, the functional and clinical significance of these 19 polymorphisms were not further pursued given the negative findings from the functional work. The PSA AR enhancer region was also assessed for potential polymorphisms that may be associated with prostate cancer risk. A total of 12 polymorphisms were identified in the PSA enhancer of which two (A-4643G and T-5412C) have been reported to alter functionality of the enhancer region and thus, prioritised for further analysis. Association analysis for prostate cancer risk was then carried out on these PSA enhancer polymorphisms as none of the KLK4 promoter polymorphisms were found in functional HREs. No significant association for either the A-4643G or T-5412C polymorphism with prostate cancer risk was found at the P = 0.05 level. However, under an age-adjusted dominant model a 1.22- (95% CI = 1.16-1.26) and 1.23-fold (95% CI = 1.17-1.29) increased risk for prostate cancer was found for the A-4643G or T-5412C polymorphisms, respectively. Both polymorphisms were also assessed for association with tumour grade and stage and PSA levels. Genotypes were significantly different for the A-4643G and T-5412C polymorphisms with tumour stage and PSA levels, respectively. However, these results are likely to be biased by the case population which consist primarily of men who presented with incidental (pT1) and organ-confined (pT2) tumours. To summarise, the A-4643G and T-5412C polymorphisms are unlikely to be associated with prostate cancer risk, PSA levels or stage/grade of disease. However, further analyses in a larger cohort is warranted given that these polymorphisms alter androgen responsiveness of the PSA enhancer and that elevated PSA levels are indicative of men with prostate cancer. To summarise, this PhD has elucidated the functional significance of the PSA G-158A polymorphism in prostate cancer and which may be important in prostate cancer patho-physiology. This PhD has also furthered the understanding of the hormonal regulation of KLK4 in prostate cancer cells. Finally, this PhD has carried out a pilot study on two functional PSA enhancer polymorphisms (A-4643G and T-5412C) with prostate cancer risk.
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Hofmann, Peter Josef. "Aufbau eines Reportergenassays zur Untersuchung der Wechselwirkung endokriner Disruptoren mit der T 3-regulierten Transaktivierung." Doctoral thesis, Humboldt-Universität zu Berlin, Mathematisch-Naturwissenschaftliche Fakultät I, 2008. http://dx.doi.org/10.18452/15813.

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Das Schilddrüsenhormon Triiodthyronin (T3) ist ein essenzieller Regulator physiologischer Prozesse der Entwicklung, des Wachstums und im Intermediärstoffwechsel. Täglich werden zahlreiche natürliche und synthetische Stoffe aufgenommen, die mit dem endokrinen System interferieren und deshalb als Endokrine Disruptoren (ED) bezeichnet werden. Zur Untersuchung einer direkten Interferenz von ED mit den Schilddrüsenhormonrezeptoren (TR) und ihrer transkriptionellen Aktivität wurde im Rahmen dieser Arbeit ein neues Luziferase-basiertes T3 Reportergensystem mit TRalpha1-transfizierten humanen Leberzellen aufgebaut. Durch Validierung mit dem synthetischen TR-Agonisten GC-1 und dem Antagonisten NH-3 konnte nachgewiesen werden, dass dieser Assay ein hoch-sensitives System zur Analyse von agonistischen sowie antagonistischen Effekten von Testsubtanzen darstellt. Zur Bestimmung der endokrinen Aktivitäten einiger humanrelevanter Vertreter aus den Stoffkategorien der Nahrungsmittel, Kosmetika, Pestizide und Industriechemikalien wurden Dosis-Wirkungskurven in Aktivierungs- und T3-Kompetitionsexperimenten ermittelt. In mikromolaren Konzentrationen wirkten von insgesamt 21 Testsubstanzen einige als reine Agonisten oder Antagonisten während andere gemischt agonistische/antagonistische Effekte hatten. Aufgrund ihrer hier beobachteten Effekte und der gegebenen Humanexposition wird eine eingehendere Analyse von 4-Methylbenzyliden Campher, 4-Nonylphenol, Acetochlor, Benzophenon 2, Benzophenon 3, Bisphenol A, Genistein, Octylmethoxycinnamat, Tetrabromobisphenol A und Xanthohumol empfohlen. Außerdem erwiesen sich einige Metaboliten von Schilddrüsenhormonen als potente Agonisten im T3-Reportergenassay und bedürfen weiterer Aufmerksamkeit. Für die molekulare Charakterisierung der Einflüsse solcher Substanzen auf die T3-regulierte Transaktivierung konnte mit dem hier etablierten Bioassay ein zuverlässiges neues Testsystem für reproduzierbare Screeningserien geschaffen werden.<br>Triiodothyronine (T3) is a crucial regulator of many physiological processes during development, growth and metabolism. A variety of natural and synthetic substances, which are collectively termed endocrine disrupters (ED) due to their interference with the endocrine system, is taken up on a daily base. A novel luciferase-based T3-responsive reporter gene system employing a human liver cell line transfected with thyroid hormone receptor (TR) alpha1 was established in this work to elucidate the potential molecular interference of certain ED with TR and their transcriptional activity. This assay was validated to be a highly sensitive and reliable tool for analyzing agonistic and antagonistic effects of test compounds using the synthetic TR agonist GC-1 and the antagonist NH-3. Dose-response data of test compounds contained in food, cosmetics, pesticides, plasticizers and other industrial chemicals were obtained after applying the substances alone in activation assays or in combination with T3 in competition assays. In total 21 test compounds were screened of which some acted as pure agonists or antagonists while others were mixed agonists/antagonists in the micromolar concentration range and only one was without effect. Follow-up studies are recommended for some of these substances with regard to their effects as determined in this bioassay and in light of information known on human exposure, i.e., 4-methylbenzyliden camphor, 4-nonylphenol, acetochlor, benzophenone 2, benzophenone 3, bisphenol A, genistein, octylmethoxycinnamate, tetrabromobisphenol A and xanthohumol. In addition some endogenous metabolites of thyroid hormones were surprisingly potent agonists in the T3 reporter gene assay and merit further attention. The novel bioassay established here represents a reliable tool for the screening and molecular characterization of substances interfering with T3-mediated transactivation of gene expression.
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Dukorn, Stefanie [Verfasser], та A. [Akademischer Betreuer] Buschauer. "Pharmacological tools for the NPY receptors: [³⁵S]GTPγS binding assays, luciferase gene reporter assays and labeled peptides / Stefanie Dukorn ; Betreuer: A. Buschauer". Regensburg : Universitätsbibliothek Regensburg, 2018. http://d-nb.info/115170007X/34.

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Douvris, Adrianna. "Functional Analysis of the TRIB1 Locus in Coronary Artery Disease." Thèse, Université d'Ottawa / University of Ottawa, 2011. http://hdl.handle.net/10393/20115.

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The TRIB1 locus (8q24.13) is a novel locus associated with plasma TGs and CAD risk. Trib1 is a regulator of MAPK activity, and has been shown to regulate hepatic lipogenesis and VLDL production in mice. However, the functional relationship between common SNPs at the TRIB1 locus and plasma lipid traits is unknown; TRIB1 has not been identified as an eQTL. This cluster of SNPs falls within an intergenic region 25kb to 50kb downstream of the TRIB1 coding region. By phylogenetic footprinting analysis and DNA genotyping, we identified an evolutionarily conserved region (CNS1) within the risk locus that harbours two common SNPs in tight LD with GWAS risk SNPs and significantly associated with CAD. We investigated the regulatory function of CNS1 by luciferase reporter assays in HepG2 cells and demonstrate that this region has promoter activity. In addition, the rs2001844 risk allele significantly reduces luciferase activity, suggesting that altered expression of the EST-based gene may be associated with plasma TGs. We identified an EST within the risk locus directly downstream of CNS1. We performed 5'/3' RACE using HepG2 RNA, identified multiple variants of this EST-based gene, and confirmed its transcription start site within CNS1. We hypothesize that this EST is a long noncoding RNA due to low abundance, poor conservation, and absence of significant ORF. Over-expression of a short variant implicates its function in the regulation of target gene transcription, although the mechanism of action remains unknown. We conclude that the risk locus at 8q24.13 harbours a novel EST-based gene that may explain the relationship between GWAS SNPs at this locus and plasma lipid traits.
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Lu, Yu-Jou, and 陸羽柔. "Analysis of HHP1 Promoter Using Luciferase Reporter Assay System." Thesis, 2011. http://ndltd.ncl.edu.tw/handle/54762340551517489882.

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碩士<br>國立臺灣大學<br>生化科技學系<br>99<br>HHP1 (heptahelical transmembrane protein 1), a protein with a predicted seven transmembrane domain structure homologous to mPRs (membrane progestin receptors), is determined to be a novel negative regulator in ABA and osmotic signaling. Sequence analysis showed that the fragment upstream to the coding region of HHP1 (5’ UTR + -1 ~ -1552 bp) contains many ABA-, dehydration-, salinity- and low temperature-responsive elements. In this study, we tried to investigate the stress-responsive elements which are involved in HHP1 transcriptional regulation using Dual-Luciferase Reporter○R (DLRTM ) Assay System (Promega). Four different DNA fragments containing various length of the putative promoter region of HHP1 (U1, 5’ UTR + -1 ~ -1552 bp; U2, 5’ UTR + -1 ~ -1103 bp; U3, 5’ UTR + -1 ~ -740 bp and U4, 5’ UTR + -1 ~ -302 bp) were cloned into vectors harboring firefly luciferase (FLUC). These constructs (U1 ~ U4-pSP) were co-transfected into protoplasts isolated from Arabidopsis mesophyll with internal control plasmid 35S-pRL expressing Renilla luciferase (RLUC). Due to the methods used to isolate and transfect plasmid into protoplasts greatly affected the assay, a few modification were made to optimize the protoplasts isolation and transfection. Protoplasts isolated from 5 ~ 7 week-old Arabidopsis, 2 × 106 cells/MMG solution for transfection, and 100:1 mass ratio of U1 ~ U4-pSP to 35S-pRL gave the best transformation and normalized luciferase expression (FLUC/RLUC). From more than three independent experiments, higher luciferase expressions were observed in the U4-pSP construct which contains the shorter HHP1 upstream fragment. The experiments were also performed when the protoplasts were treated with abscisic acid. A few interesting preliminary results were discussed.
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Lin, Yun-Tong, and 林筠彤. "The small RNA of Japanese encephalitis virus inhibits translation efficiency analyzed by a luciferase reporter gene assay." Thesis, 2008. http://ndltd.ncl.edu.tw/handle/82w7a2.

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碩士<br>國立東華大學<br>生物技術研究所<br>96<br>Japanese encephalitis virus (JEV) contains a single positive-strand RNA genome of 10,976 nucleotides in length. The 5’- and 3’-terminal regions contain conserved and highly ordered secondary structures that have been shown to play important roles in viral translation and replication. Previous studies in our laboratory have shown the abundant accumulation of a 3’-terminal 521- to 523-nucleotide genome fragment, representing the highly conserved region of the 3’-untranslated region, in JEV-infected cells. Function of the small RNA remains unclear. The small RNA was detected in Dengue Type 2 virus-infected cells in this study suggesting the small RNA may exist among all flaviviruses. The molar ratio of the small RNA to the genome is greater in JEV persistently-infected cells (3.36; survival cells from acutely infection, approximately 100 days postinfection) than those in JEV acutely-infected cells (0.93; 2 days postinfection prior to cell death) indicated that the small RNA plays a role in establishment of persistent infection. A small portion of viral genomic RNA was found in the nuclear fraction, while the small RNA was only detected in the cytoplasmic fraction. An RNA interference detection assay was used to address possible effect of the small RNA in this cellular RNA degradation pathway but the results showed no significant effect. Surprisingly, the small RNA alone inhibits the translation efficiently of the firefly luciferase. In addition, a Renilla luciferase (Rluc) gene reporter in-frame fused with the 5’ and 3’ terminal sequences of JEV resulting as a JEV minicon and from this, the synthetic RNA transcript was made for viral translation assay. This JEV minicon was proved to be cap-dependent translation. Transfecting of sense- or anti-sense small RNA both inhibit JEV minicon translation efficiency, suggesting that the small RNA plays a role in viral translation.
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Pohlová, Lenka. "Onkogenní promotor c-myc jako cíl pro nový typ heterocyklických dikationtů stabilizujících G-kvadruplex." Master's thesis, 2015. http://www.nusl.cz/ntk/nusl-353836.

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Targeting oncogene promoters: a novel heterocyclic cations as G-quadruplex stabilizing ligands Lenka Pohlová Abstract: The diploma thesis studies an effect of newly synthesized group of compounds - helquats - on the expression of c-myc as a major player in malignant transformation and tumorigenesis via the stabilization of G-quadruplex in c-myc promotor. The G-quadruplex c-myc stabilization ability was tested for 101 helquats using dual luciferase reporter assay. The G-quadruplex c-myc stabilization ability was found for 13 helquats by this method. 8 successful helquats was selected by a comparison of the results from dual luciferase reporter assay and FRET melting assay. Effect on cell viability of tumor (HeLa S3) and non-tumor (HUVEC) cell lines was evaluated for these 8 helquats. Three of them exhibited cytotoxic effect on tumor cells but no effect was observed on viability of non-tumor cells. Moreover, an effect of these 3 helquats on c-myc expression on both mRNA and protein level, where significant effect on c-myc mRNA expression was not found for most of incubation periods. The 30% decrease in mRNA level was observed only for 24 hours incubation period for two helquats (LS702 and MJ656). The decrease in the expression on protein level was observed for all tested helquats, and helquat LS702 had the...
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Kožantová, Jana. "Měření aktivace signálních drah v myší makrofágové linii IC-21 a primárních dendritických buňkách po infekci virem klíšťové encefalitidy." Master's thesis, 2017. http://www.nusl.cz/ntk/nusl-367677.

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Tick-borne encephalitis is a serious disease of the central nervous system. It is caused by tick-borne encephalitis virus, which is transmitted by ticks. The Czech Republic is one of the countries with the highest prevalence of this disease. Tick-borne encephalitis virus is able to replicate in several cell types. In this work we focused on macrophage line IC-21 and dendritic cells, because these cells are the first, which encounter the virus and support its spreading in the host at early stage of infection. So far there is not known any specific receptor for virus entry into cells or which signaling pathways activates. Therefore, we decided to investigate the activation of selected signaling pathways after infection with tick-borne encephalitis virus and influence of tick saliva on this activation. We employed methods of dual luciferase reporter assay, immunosandwich assay and western blot. The obtained results showed that in virus infected IC-21 cells are activated phosphatidyl-inositol pathway, NF-κB pathway, signaling molecule Erk1/2 and others. Testing of tick saliva effect revealed significantly decreased activity of NF-κB, AP-1 and CREB.
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Book chapters on the topic "Reporter luciferase assay"

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Wettey, Frank R., and Antony P. Jackson. "Luciferase reporter assay." In Subcellular Biochemistry. Springer Netherlands, 2006. http://dx.doi.org/10.1007/978-1-4020-4896-8_39.

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Tomasello, Luisa, Landon Cluts, and Carlo M. Croce. "Experimental Validation of MicroRNA Targets: Luciferase Reporter Assay." In Methods in Molecular Biology. Springer New York, 2019. http://dx.doi.org/10.1007/978-1-4939-9207-2_17.

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Montefiori, David C. "Measuring HIV Neutralization in a Luciferase Reporter Gene Assay." In Methods in Molecular Biology. Humana Press, 2009. http://dx.doi.org/10.1007/978-1-59745-170-3_26.

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Jin, Yi, Zujian Chen, Xiqiang Liu, and Xiaofeng Zhou. "Evaluating the MicroRNA Targeting Sites by Luciferase Reporter Gene Assay." In MicroRNA Protocols. Humana Press, 2012. http://dx.doi.org/10.1007/978-1-62703-083-0_10.

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Marcos-Vadillo, Elena, and Asunción García-Sánchez. "Promoter Assay Using Luciferase Reporter Gene in the A549 Cell Line." In Methods in Molecular Biology. Springer New York, 2016. http://dx.doi.org/10.1007/978-1-4939-3652-6_14.

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Iglesias-Ara, Ainhoa, Nerea Osinalde, and Ana M. Zubiaga. "Detection of E2F-Induced Transcriptional Activity Using a Dual Luciferase Reporter Assay." In Methods in Molecular Biology. Springer New York, 2018. http://dx.doi.org/10.1007/978-1-4939-7565-5_14.

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Vincelli, Gabriele, and Andrea Bedini. "Renilla Luciferase Reporter Assay to Study 3′UTR-Driven Posttranscriptional Regulations of OPRM1." In Methods in Molecular Biology. Springer New York, 2014. http://dx.doi.org/10.1007/978-1-4939-1708-2_4.

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Müller, Katharina, Manfred Ogris, and Haider Sami. "Firefly Luciferase-Based Reporter Gene Assay for Investigating Nanoparticle-Mediated Nucleic Acid Delivery." In Nanotechnology for Nucleic Acid Delivery. Springer New York, 2019. http://dx.doi.org/10.1007/978-1-4939-9092-4_15.

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Vincelli, Gabriele, and Andrea Bedini. "Renilla Luciferase Reporter Assay to Study 3′-UTR-Driven Posttranscriptional Regulation of OPRM1." In Methods in Molecular Biology. Springer US, 2020. http://dx.doi.org/10.1007/978-1-0716-0884-5_2.

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Lieber, Diana, and Susanne M. Bailer. "Determination of HSV-1 Infectivity by Plaque Assay and a Luciferase Reporter Cell Line." In Methods in Molecular Biology. Humana Press, 2013. http://dx.doi.org/10.1007/978-1-62703-601-6_12.

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Conference papers on the topic "Reporter luciferase assay"

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Shi, Yihui, Jaehyeon Park, Chandraiah Lagisetti, Wei Zhou, Lidia C. Sambucetti, and Thomas R. Webb. "Abstract 4193: A triple exon-skipping luciferase reporter assay identifies a new CLK inhibitor scaffold." In Proceedings: AACR Annual Meeting 2017; April 1-5, 2017; Washington, DC. American Association for Cancer Research, 2017. http://dx.doi.org/10.1158/1538-7445.am2017-4193.

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Fields, Wanda R., Wolfgang Liedtke, Jinju Li, and Betsy Bombick. "Abstract 1693: Assessment of Nrf2 luciferase reporter assay and heme oxygenase expression as models of oxidative stress inin vitrocultures of lung cells exposed to cigarette smoke condensate." In Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC. American Association for Cancer Research, 2010. http://dx.doi.org/10.1158/1538-7445.am10-1693.

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Liu, Haiyun, Brian Della Fera, John Foulke, Luping Chen, and Fang Tian. "Abstract 1306: Primary NK cells and luciferase expressing reporter cell lines for use in developing ADCC assays for immuno-oncology drug screening." In Proceedings: AACR Annual Meeting 2021; April 10-15, 2021 and May 17-21, 2021; Philadelphia, PA. American Association for Cancer Research, 2021. http://dx.doi.org/10.1158/1538-7445.am2021-1306.

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