scholarly journals Evaluation of Firefly and Renilla Luciferase Inhibition in Reporter-Gene Assays: A Case of Isoflavonoids

2021 ◽  
Vol 22 (13) ◽  
pp. 6927
Author(s):  
Maša Kenda ◽  
Jan Vegelj ◽  
Barbara Herlah ◽  
Andrej Perdih ◽  
Přemysl Mladěnka ◽  
...  
Keyword(s):  
Reporter Gene ◽  
Firefly Luciferase ◽  
Qsar Model ◽  
In Vitro Assay ◽  
Biochanin A ◽  
Renilla Luciferase ◽  
Luciferase Reporter ◽  
Vitro Assay ◽  
Reporter Gene Assays

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.

scholarly journals In vivo analysis of the myosin heavy chain IIB promoter region

1998 ◽  
Vol 274 (3) ◽  
pp. C681-C687 ◽  
Author(s):  
Steven J. Swoap
Keyword(s):  
Reporter Gene ◽  
Luciferase Activity ◽  
Fiber Type ◽  
Firefly Luciferase ◽  
Renilla Luciferase ◽  
Luciferase Reporter ◽  
Base Pairs ◽  
Specific Expression ◽  
Mhc Iib

The myosin heavy chain (MHC) IIB gene is preferentially expressed in fast-twitch muscles of the hindlimb, such as the tibialis anterior (TA). The molecular mechanism(s) for this preferential expression are unknown. The goals of the current study were 1) to determine whether the cloned region of the MHC IIB promoter contains the necessary cis-acting element(s) to drive fiber-type-specific expression of this gene in vivo, 2) to determine which region within the promoter is responsible for fiber-type-specific expression, and 3) to determine whether transcription off of the cloned region of the MHC IIB promoter accurately mimics endogenous gene expression in a muscle undergoing a fiber-type transition. To accomplish these goals, a 2.6-kilobase fragment of the promoter-enhancer region of the MHC IIB gene was cloned upstream of the firefly luciferase reporter gene and coinjected with pRL-cytomegalovirus (CMV) (CMV promoter driving the renilla luciferase reporter) into the TA and the slow soleus muscle. Firefly luciferase activity relative to renilla luciferase activity within the TA was 35-fold greater than within the soleus. Deletional analysis demonstrated that only the proximal 295 base pairs (pGL3IIB0.3) were required to maintain this muscle-fiber-type specificity. Reporter gene expression of pGL3IIB0.3 construct was significantly upregulated twofold in unweighted soleus muscles compared with normal soleus muscles. Thus the region within the proximal 295 base pairs of the MHC IIB gene contains at least one element that can drive fiber-type-specific expression of a reporter gene.

scholarly journals Rational Design of a Triple Reporter Gene for Multimodality Molecular Imaging

2014 ◽  
Vol 2014 ◽  
pp. 1-11
Author(s):  
Ya-Ju Hsieh ◽  
Luen Hwu ◽  
Chien-Chih Ke ◽  
Skye Hsin-Hsien Yeh ◽  
Chien-Feng Lin ◽  
...  
Keyword(s):  
Reporter Gene ◽  
Rational Design ◽  
Multimodality Imaging ◽  
Firefly Luciferase ◽  
Luciferase Reporter ◽  
Fluorescence Signal ◽  
Red Fluorescence ◽  
Simplex Virus

Multimodality imaging using noncytotoxic triple fusion (TF) reporter genes is an important application for cell-based tracking, drug screening, and therapy. The firefly luciferase(fl), monomeric red fluorescence protein(mrfp), and truncated herpes simplex virus type 1 thymidine kinase SR39 mutant(ttksr39)were fused together to create TF reporter gene constructs with different order. The enzymatic activities of TF protein in vitro andin vivowere determined by luciferase reporter assay,H-FEAU cellular uptake experiment, bioluminescence imaging, and micropositron emission tomography (microPET). The TF construct expressed in H1299 cells possesses luciferase activity and red fluorescence. The tTKSR39 activity is preserved in TF protein and mediates high levels ofH-FEAU accumulation and significant cell death from ganciclovir (GCV) prodrug activation. In living animals, the luciferase and tTKSR39 activities of TF protein have also been successfully validated by multimodality imaging systems. The red fluorescence signal is relatively weak forin vivoimaging but may expedite FACS-based selection of TF reporter expressing cells. We have developed an optimized triple fusion reporter constructDsRedm-fl-ttksr39for more effective and sensitivein vivoanimal imaging using fluorescence, bioluminescence, and PET imaging modalities, which may facilitate different fields of biomedical research and applications.

Novel in vitro assay for the detection of pharmacologic inducers of fetal hemoglobin

Blood ◽  
2000 ◽  
Vol 96 (1) ◽  
pp. 321-326 ◽  
Author(s):  
Evangelia Skarpidi ◽  
George Vassilopoulos ◽  
Qiliang Li ◽  
George Stamatoyannopoulos
Keyword(s):  
Luciferase Activity ◽  
Globin Gene ◽  
Fetal Hemoglobin ◽  
Reporter Genes ◽  
Firefly Luciferase ◽  
Gene Activity ◽  
Renilla Luciferase ◽  
Luciferase Reporter ◽  
Vitro Assay ◽  
Highly Sensitive

Current techniques for identifying fetal hemoglobin (HbF) inducers are complex and time consuming. We developed a rapid and efficient method for detecting HbF inducers. Our system uses a recombinant DNA construct in which the coding sequences of 2 different luciferase reporter genes, firefly and renilla, are substituted for those of human γ and β globin genes, respectively. The activity of these genes can be distinguished by a simple, highly sensitive enzymatic assay in cell lysates. GM979 cells stably transfected with the construct are cultured in the presence of compounds, and their effects are determined by measuring the changes in activity of the 2 luciferase genes. Specific γ globin gene inducers are recognized by their ability to increase γ-firefly luciferase (γF) gene activity significantly more than β-renilla luciferase (βR) gene activity, identified by an increased ratio of γ-firefly luciferase activity over total luciferase activity. These results suggest that the use of the 2 luciferase reporter genes provides a simple, highly sensitive, and reproducible system for the detection of compounds that increase γ-globin gene expression. It can therefore be used for the screening of chemical agents that may have γ-globin gene inducibility.

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.

Structural and functional effects of circular permutation on firefly luciferase: In vitro assay of caspase 3/7

2013 ◽  
Vol 58 ◽  
pp. 336-342 ◽  
Author(s):  
Roya Cheraghi ◽  
Saman Hosseinkhani ◽  
Jamshid Davoodi ◽  
Mahboobeh Nazari ◽  
Zahra Amini-Bayat ◽  
...  
Keyword(s):  
Caspase 3 ◽  
Firefly Luciferase ◽  
Circular Permutation ◽  
In Vitro Assay ◽  
Vitro Assay

Quenching the firefly bioluminescence by various ions

2016 ◽  
Vol 15 (2) ◽  
pp. 244-249 ◽  
Author(s):  
Huateng Zhang ◽  
Haixiu Bai ◽  
Tianyu Jiang ◽  
Zhao Ma ◽  
Yanna Cheng ◽  
...  
Keyword(s):  
Reporter Gene ◽  
Firefly Luciferase ◽  
Reporter Gene Assay ◽  
Negligible Effect ◽  
Renilla Luciferase ◽  
Luciferase Reporter ◽  
Luciferase Reporter Gene ◽  
Luciferase Reporter Gene Assay ◽  
Gene Assay ◽  
Firefly Bioluminescence

Some specific ions could selectively inhibit firefly luciferase while having a negligible effect on renilla luciferase, which may be used in the improved dual luciferase reporter gene assay.

scholarly journals Identification of Inhibitors of Bacterial Transcription/Translation Machinery Utilizing a Miniaturized 1536-Well Format Screen

2001 ◽  
Vol 6 (4) ◽  
pp. 233-243 ◽  
Author(s):  
Ilona Kariv ◽  
Hong Cao ◽  
Phillip D. Marvil ◽  
Ekaterina V. Bobkova ◽  
Yuri E. Bukhtiyarov ◽  
...  
Keyword(s):  
Multicomponent System ◽  
Firefly Luciferase ◽  
Luciferase Reporter ◽  
Luciferase Reporter Gene ◽  
Data Set ◽  
Vitro Assay ◽  
Bacterial Transcription ◽  
Firefly Luciferase Reporter ◽  
Firefly Luciferase Reporter Gene

This report presents the miniaturization of a HTS screen to identify inhibitors of prokaryotic transcription-translation in a 1536-well format. The in vitro assay design utilized the bacterial expression machinery to drive expression of a firefly luciferase reporter gene, which was read as an endpoint luminesence measurement. This multicomponent system permits identification of inhibitors at different steps in this pathway. Successful miniaturization required integration of homogeneous assay formats, robust liquid-handling workstations, and second-generation imaging systems. Comparison of data from a triplicate 1536-well screen of a subset of a target library that had been previously validated and followed up for hit confirmation in a 384-well plate format confirmed that triplicate screening yields data of higher confidence and quality, eliminates the time-consuming and potentially error-prone step of cherry-picking, and reduces the number of false positives and negatives. The substantial savings of reagents and reduction of the numbers of plates to process obtained in a 1536-well format as compared to a 384-well format allowed a full triplicate evaluation of the entire library of 183,000 compounds at lower cost and in less time. The triplicate-screen statistics are consistent with a highly reliable data set with a coefficient of variation of 14.8% and Z' and Z values of 0.57 and 0.25, respectively. This screen resulted in the identification of 1,149 hits (0.63% hit rate), representing a compound population at 2.5 standard deviations from the mean cutoff. Furthermore, the data demonstrate good agreement between IC50 values derived for this assay in a 1536-well format and 384-well format.

Novel in vitro assay for the detection of pharmacologic inducers of fetal hemoglobin

Blood ◽  
2000 ◽  
Vol 96 (1) ◽  
pp. 321-326 ◽  
Author(s):  
Evangelia Skarpidi ◽  
George Vassilopoulos ◽  
Qiliang Li ◽  
George Stamatoyannopoulos
Keyword(s):  
Luciferase Activity ◽  
Globin Gene ◽  
Fetal Hemoglobin ◽  
Reporter Genes ◽  
Firefly Luciferase ◽  
Gene Activity ◽  
Renilla Luciferase ◽  
Luciferase Reporter ◽  
Vitro Assay ◽  
Highly Sensitive

Abstract Current techniques for identifying fetal hemoglobin (HbF) inducers are complex and time consuming. We developed a rapid and efficient method for detecting HbF inducers. Our system uses a recombinant DNA construct in which the coding sequences of 2 different luciferase reporter genes, firefly and renilla, are substituted for those of human γ and β globin genes, respectively. The activity of these genes can be distinguished by a simple, highly sensitive enzymatic assay in cell lysates. GM979 cells stably transfected with the construct are cultured in the presence of compounds, and their effects are determined by measuring the changes in activity of the 2 luciferase genes. Specific γ globin gene inducers are recognized by their ability to increase γ-firefly luciferase (γF) gene activity significantly more than β-renilla luciferase (βR) gene activity, identified by an increased ratio of γ-firefly luciferase activity over total luciferase activity. These results suggest that the use of the 2 luciferase reporter genes provides a simple, highly sensitive, and reproducible system for the detection of compounds that increase γ-globin gene expression. It can therefore be used for the screening of chemical agents that may have γ-globin gene inducibility.

scholarly journals Hsa_circ_0011385 knockdown represses cell proliferation in hepatocellular carcinoma

2021 ◽  
Vol 7 (1) ◽  
Author(s):  
Chuangye Ni ◽  
Shikun Yang ◽  
Yang Ji ◽  
Yunfei Duan ◽  
Wenjie Yang ◽  
...  
Keyword(s):  
Hepatocellular Carcinoma ◽  
Cell Proliferation ◽  
Cell Lines ◽  
Reporter Gene ◽  
Luciferase Reporter ◽  
Mrna Levels ◽  
Luciferase Reporter Gene ◽  
Reporter Gene Assays

AbstractCircular RNAs (circRNAs), continuous loops of single-stranded RNA, regulate gene expression during the development of various cancers. However, the function of circRNAs in hepatocellular carcinoma (HCC) is rarely discussed. Quantitative real-time polymerase chain reaction (qRT-PCR) was used to determine the mRNA levels of circ_0011385, miR-361-3p, and STC2 in 96 pairs of HCC tissues (tumor tissues and adjacent normal tissues), HCC cell lines, and L02 (human normal liver cell line) cells. The relationships between circ_0011385 expression and clinical features of HCC were evaluated. Functional experiments in vitro or in vivo were used to evaluate the biological function of circ_0011385. Bioinformatics analysis was performed to predict miRNAs and mRNAs sponged by circ_0011385. RNA immunoprecipitation (RIP) and dual-luciferase reporter gene assays were used to elucidate the interactions among circ_0011385, miR-361-3p, and STC2 (stanniocalcin 2). ChIP and dual-luciferase reporter gene assays were used to identify the upstream regulator of circ_0011385. High expression of circ_0011385 was observed in HCC tissues and cell lines and was significantly associated with tumor size, TNM stage, and prognosis. In addition, inhibition of circ_0011385 expression prevented the proliferation of HCC cells in vitro and in vivo. Circ_0011385 sponged miR-361-3p, thereby regulating the mRNA expression of STC2. In addition, the transcription of circ_0011385 was regulated by SP3. Circ_0011385 knockdown suppressed cell proliferation and tumor activity in HCC. Circ_0011385 may therefore serve as a new biomarker in the diagnosis and treatment of HCC.

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