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.

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 Use of Leishmania donovani Field Isolates Expressing the Luciferase Reporter Gene in In Vitro Drug Screening

2005 ◽  
Vol 49 (9) ◽  
pp. 3776-3783 ◽  
Author(s):  
Ashutosh ◽  
Suman Gupta ◽  
Ramesh ◽  
Shyam Sundar ◽  
Neena Goyal
Keyword(s):  
Reporter Gene ◽  
High Throughput ◽  
High Throughput Screening ◽  
Leishmania Donovani ◽  
Luciferase Activity ◽  
Firefly Luciferase ◽  
Luciferase Reporter ◽  
Luciferase Reporter Gene ◽  
Field Isolates

ABSTRACT Currently available primary screens for the selection of candidate antileishmanial compounds are not ideal. These techniques are time-consuming, laborious, and difficult to scale and require macrophages, which limit their use for high-throughput screening. We have developed Leishmania donovani field isolates that constitutively express the firefly luciferase reporter gene (luc) as a part of an episomal vector. An excellent correlation between parasite number and luciferase activity was observed. luc expression was stable, even in the absence of drug selection, for 4 weeks. The transfectants were infective to macrophages, and intracellular amastigotes exhibited luciferase activity. The suitability of these recombinant field isolates for in vitro screening of antileishmanial drugs was established. The luciferase-expressing sodium stibogluconate-resistant cell lines offer a model for the screening of compounds for resistance. The system is in routine use at the Central Drug Research Institute, Lucknow, India, for high-throughput screening of newly synthesized compounds.

scholarly journals Hormonal regulation of the human sterol 27-hydroxylase gene CYP27A1

2003 ◽  
Vol 372 (2) ◽  
pp. 529-534 ◽  
Author(s):  
Zufan ARAYA ◽  
Wanjin TANG ◽  
Kjell WIKVALL
Keyword(s):  
Reporter Gene ◽  
Hormonal Regulation ◽  
Promoter Activity ◽  
Luciferase Activity ◽  
Luciferase Reporter ◽  
Signal Transduction Pathways ◽  
Cytochrome P450 Enzyme ◽  
Luciferase Reporter Gene ◽  
Response Elements ◽  
P450 Enzyme

The mitochondrial sterol 27-hydroxylase (CYP27A1) is a multifunctional cytochrome P450 enzyme that catalyses important hydroxylations in the biosynthesis of bile acids and bioactivation of vitamin D3. Previous results [Babiker, Andersson, Lund, Xiu, Deeb, Reshef, Leitersdorf, Diczfalusy and Björkhem (1997) J. Biol. Chem. 272, 26253–26261] suggest that CYP27A1 plays an important role in cholesterol homoeostasis and affects atherogenesis. In the present study, the regulation of the human CYP27A1 gene by growth hormone (GH), insulin-like growth factor-1 (IGF-1), dexamethasone, thyroid hormones and PMA was studied. HepG2 cells were transfected transiently with luciferase reporter gene constructs containing DNA fragments flanking the 5′-region of the human CYP27A1 gene. GH, IGF-1 and dexamethasone increased the promoter activity by 2–3-fold, whereas thyroxine (T4) and PMA repressed the activity significantly when measured with luciferase activity expressed in the cells. The endogenous CYP27A1 enzyme activity in the cells was stimulated by GH, IGF-1 and dexamethasone, whereas T4 and PMA inhibited the activity. Experiments with progressive deletion/luciferase reporter gene constructs indicated that the response elements for GH may be localized in a region upstream to position −1094 bp. The putative response elements for dexamethasone were mapped to positions between −792 and −1095 bp. The −451 bp fragment of the human CYP27A1 gene was found to confer the activation by IGF-1, and the inhibition by T4 and PMA. Results of the present study suggest that CYP27A1 is regulated in human cells by hormones and signal-transduction pathways.

scholarly journals Tissue- and time-dependent estrogen receptor activation in estrogen reporter mice

2004 ◽  
Vol 32 (3) ◽  
pp. 689-701 ◽  
Author(s):  
JG Lemmen ◽  
RJ Arends ◽  
AL van Boxtel ◽  
PT van der Saag ◽  
B van der Burg
Keyword(s):  
Reporter Gene ◽  
Imaging System ◽  
Luciferase Activity ◽  
Estrogenic Activity ◽  
Receptor Activation ◽  
Time Dependent ◽  
Luciferase Reporter ◽  
In Vivo Model ◽  
Luciferase Reporter Gene

With the aim of developing an in vivo model that directly detects activation of estrogen receptors (ERs), transgenic mice carrying a luciferase reporter gene were generated. The luciferase reporter gene was under the control of three consensus estrogen-responsive elements (EREs) coupled to a minimal TATA-box, with or without flanking chick beta-globin insulators. By using this model in combination with the IVIS imaging system, in vivo ER activation was measured. Dose- and time-dependent luciferase activity was induced in various organs of adult transgenic male mice exposed to diethylstilbestrol (DES) (10-1000 micro g/kg) and 17beta-estradiol dipropionate (EP) (10-1000 micro g/kg), when luciferase activity was measured ex vivo. The highest (>10 000-fold) induction of luciferase was measured in bone and kidney 24 h after exposure to 1000 micro g/kg EP. Other highly responsive organs include liver, testis, pituitary, brain, prostate and colon, which show different activity profiles. This in vivo model for detecting estrogenic activity can be used to assess tissue-specific action of ER agonists and antagonists. These could include selective ER modulators and environmental estrogens. In combination with the IVIS imaging system, this in vivo model is a powerful tool for assessing the kinetics of gene activation by estrogenic compounds.

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