Construction and testing of a bacterial luciferase reporter gene system forin Vivo measurement of nonsense suppression inStreptomyces

2006 ◽  
Vol 51 (1) ◽  
pp. 62-64 ◽  
Author(s):  
J. Weiser ◽  
K. Buriánková ◽  
L. Kalachová ◽  
P. Branny ◽  
J. L. Pernodet
Keyword(s):  
Reporter Gene ◽  
Nonsense Suppression ◽  
Luciferase Reporter ◽  
Luciferase Reporter Gene ◽  
Bacterial Luciferase ◽  
Gene System ◽  
Reporter Gene System

scholarly journals Analysis of Promoter Function in Aspergillus fumigatus

2012 ◽  
Vol 11 (9) ◽  
pp. 1167-1177 ◽  
Author(s):  
Sanjoy Paul ◽  
J. Stacey Klutts ◽  
W. Scott Moye-Rowley
Keyword(s):  
Aspergillus Fumigatus ◽  
Reporter Gene ◽  
Reporter Genes ◽  
Antifungal Drugs ◽  
Luciferase Reporter ◽  
Pcr Analysis ◽  
Luciferase Reporter Gene ◽  
Content Type ◽  
Gene System ◽  
Reporter Gene System

ABSTRACTThe filamentous fungusAspergillus fumigatusis an important opportunistic pathogen that can cause high mortality levels in susceptible patient populations. The increasing dependence on antifungal drugs to controlA. fumigatushas led to the inevitable acquisition of drug-resistant forms of this pathogen. In other fungal pathogens, drug resistance is often associated with an increase in transcription of genes such as ATP-binding cassette (ABC) transporters that directly lead to tolerance to commonly employed antifungal drugs. InA. fumigatus, tolerance to azole drugs (the major class of antifungal) is often associated with changes in the sequence of the azole target enzyme as well as changes in the transcription level of this gene. The target gene for azole drugs inA. fumigatusis referred to ascyp51A. In order to dissect transcription ofcyp51Atranscription and other genes of interest, we constructed a set of firefly luciferase reporter genes designed for use inA. fumigatus. These reporter genes can either replicate autonomously or be targeted to thepyrGlocus, generating an easily assayable uracil auxotrophy. We fused eight differentA. fumigatuspromoters to luciferase. Faithful behaviors of these reporter gene fusions compared to their chromosomal equivalents were evaluated by 5′ rapid amplification of cDNA ends (RACE) and quantitative reverse transcription-PCR (qRT-PCR) analysis. We used this reporter gene system to study stress-regulated transcription of a Hsp70-encoding gene, map an important promoter element in thecyp51Agene, and correct an annotation error in the actin gene. We anticipate that this luciferase reporter gene system will be broadly applicable in analyses of gene expression inA. fumigatus.

Evaluation of a CRE-Directed Luciferase Reporter Gene Assay as an Alternative to Measuring cAMP Accumulation

1997 ◽  
Vol 2 (4) ◽  
pp. 235-240 ◽  
Author(s):  
Samantha E. George ◽  
Peter J. Bungay ◽  
Louise H. Naylor
Keyword(s):  
Reporter Gene ◽  
Luciferase Reporter ◽  
Luciferase Expression ◽  
Functional Assay ◽  
Ionophore A23187 ◽  
Camp Accumulation ◽  
Luciferase Reporter Gene ◽  
Gene System ◽  
Gene Assay ◽  
Reporter Gene System

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.

Differential Effects of Components in Artemisia annua Extract on the Induction of Drug-Metabolizing Enzyme Expression Mediated by Nuclear Receptors

Planta Medica ◽  
2020 ◽  
Vol 86 (12) ◽  
pp. 867-875
Author(s):  
Xueli Zhang ◽  
Ran Meng ◽  
Haina Wang ◽  
Jie Xing
Keyword(s):  
Reporter Gene ◽  
Artemisia Annua ◽  
Constitutive Androstane Receptor ◽  
Pregnane X Receptor ◽  
Luciferase Reporter ◽  
Induction Effect ◽  
Luciferase Reporter Gene ◽  
Gene System ◽  
Drug Metabolizing Enzyme ◽  
Reporter Gene System

Abstract Artemisia annua tea is a popular dosage form used to treat and prevent malaria in some developing countries. However, repeated drinking leads to an obviously decreased efficacy, which may be related to the induction of metabolizing enzymes by artemisinin. In the present study, the ability of different components in A. annua to activate the pregnane X receptor and constitutive androstane receptor was evaluated by the dual luciferase reporter gene system. The changes in mRNA and protein expression of CYP3A4 and CYP2B6 were determined by quantitative real-time PCR and Western blotting. Results showed that in the pregnane X receptor-mediated CYP3A4 reporter gene system, chrysosplenetin and arteannuin B exhibited a weak induction effect on pregnane X receptor wt, while arteannuin A had a strong induction effect on pregnane X receptor wt and pregnane X receptor 370 and a weak induction effect on pregnane X receptor 163. In the pregnane X receptor-mediated CYP2B6 reporter gene system, arteannuin A had a moderate induction effect on pregnane X receptor wt and pregnane X receptor 379, and a weak induction effect on pregnane X receptor 403, while arteannuin B had a weak induction effect on pregnane X receptor wt and pregnane X receptor 379. Arteannuin A had a strong induction effect on constitutive androstane receptor 3 in constitutive androstane receptor-mediated CYP3A4/2B6 reporter gene systems, while arteannuin B showed a weak induction effect on constitutive androstane receptor 3 in the constitutive androstane receptor-mediated CYP2B6 reporter gene system. The mRNA and protein expressions of CYP3A4 and CYP2B6 were increased when the pregnane X receptor or constitutive androstane receptor was activated. Various components present in A. annua differentially affect the activities of pregnane X receptor isoforms and the constitutive androstane receptor, which indicates the possibility of a drug-drug interaction. This partly explains the decline in efficacy after repeated drinking of A. annua tea.

scholarly journals Evaluation of the use of the luciferase-reporter-gene system for gene-regulation studies involving cyclic AMP-elevating agents

1995 ◽  
Vol 309 (2) ◽  
pp. 385-387 ◽  
Author(s):  
O Benzakour ◽  
C Kanthou ◽  
U Dennehy ◽  
A al Haq ◽  
L P Berg ◽  
...  
Keyword(s):  
Cyclic Amp ◽  
Reporter Gene ◽  
Transcriptional Activation ◽  
Luciferase Activity ◽  
Primary Cultures ◽  
Luciferase Reporter ◽  
Luciferase Reporter Gene ◽  
Promoter Elements ◽  
Gene System ◽  
Reporter Gene System

The effects of cyclic AMP (cAMP)-elevating agents on the activity of cis-acting gene promoter sequences are frequently studied using the luciferase-reporter-gene system. The aim of the present study was to assess whether cAMP-elevating agents induce any changes in the level of luciferase activity independently of a transcriptional activation of promoter elements. Chloramphenicol acteyltransferase (CAT) and luciferase reporter genes under the control of the same promoter elements were transiently expressed in primary cultures of human vascular smooth-muscle cells. Transfected cells were treated with a cell-permeable and non-hydrolysable cAMP analogue, 2′-O-monobutyryl-8-bromo cAMP, or with the cAMP-elevating agents forskolin and prostaglandin E1 (PGE1). Forskolin and PGE1 induced a significant increase in the level of luciferase activity, but had no effect on CAT activity. Conclusions based solely on the use of the luciferase-reporter-gene system in studies involving promoter activation by cAMP-elevating agents could therefore be misleading.

A sensitive reporter gene system using bacterial luciferase based on a series of plasmid cloning vectors compatible with derivatives of pBR322

Gene ◽  
1997 ◽  
Vol 186 (2) ◽  
pp. 197-200 ◽  
Author(s):  
Danielle Manen ◽  
Maryse Pougeon ◽  
Pascal Damay ◽  
Johannes Geiselmann
Keyword(s):  
Reporter Gene ◽  
Bacterial Luciferase ◽  
Cloning Vectors ◽  
Gene System ◽  
Reporter Gene System ◽  
Derivatives Of

scholarly journals Study on the Mechanism of Mirna-21 Affecting the Differentiation of Bone Marrow Mesenchymal Stem Cells into Cardiomyocyte-Like Cells By Targeting Ajuba / Isl1 Axis

2021 ◽  
Author(s):  
Wengang Yang ◽  
Song Xue ◽  
Hui Zheng ◽  
Jianggui Dan ◽  
Lei Zhao ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Mesenchymal Stem Cells ◽  
Reporter Gene ◽  
Luciferase Reporter ◽  
Myoid Cells ◽  
Luciferase Reporter Gene ◽  
Slow Virus ◽  
Marrow Mesenchymal Stem Cells ◽  
Reporter Gene System

Abstract Purpose: To study the mechanism of miRNA-21 targeting ajuba/ Isl1 axis to affect BMSC differentiation to cardiac myoid cells. Methods: BMSC was cultured and miRNA-21 was constructed to infect BMSC. The miRNA-21 was directly regulated by luciferase reporter gene system. The expression of cTnI, ajuba and Isl1 was detected by RT-qPCR and WB. The expression of cTnI, ajuba and Isl1 was detected by RT-qPCR and WB, and miR-21 was detected by RT-qPCR; The differentiation ability of BMSC in all groups was detected by RT-qPCR and WB; To evaluate the effect of ajuba and Isl1 on the differentiation ability of BMSC. Results: BMSC was cultured successfully, BMSC was successfully constructed by mir-21-OE, mir-21-KD, ajuba-OE and ajuba KD slow virus; RT-qPCR and WB were used to detect the high expression of cTnI in mir-21-OE and ajuba KD groups, and the expression of miR-21 was increased, the expression of ajuba was inhibited and Isl1 expression was enhanced. Conclusion: The expression of ajuba was inhibited by up regulation of miRNA-21 target, and the expression of Isl1 enhanced to promote BMSC differentiation, that is, miRNA-21 regulated the axis of ajuba/ Isl1 to influence the differentiation of BMSC to cardiomyocyte-like cells.

Miniaturization of a Mammalian Cell-Based Assay: Luciferase Reporter Gene Readout in a 3 Microliter 1536-Well Plate

1999 ◽  
Vol 4 (3) ◽  
pp. 137-142 ◽  
Author(s):  
Anthony M. Maffia ◽  
Ilona Kariv ◽  
Kevin R. Oldenburg
Keyword(s):  
Reporter Gene ◽  
Mammalian Cell ◽  
Stress Responses ◽  
Mammalian Cells ◽  
Chemical Diversity ◽  
Luciferase Reporter ◽  
Luciferase Reporter Gene ◽  
Microtiter Plates ◽  
Human T Cell ◽  
Reporter Gene System

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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