scholarly journals Safe and Sensitive Antiviral Screening Platform Based on Recombinant Human Coronavirus OC43 Expressing the Luciferase Reporter Gene

2016 ◽  
Vol 60 (9) ◽  
pp. 5492-5503 ◽  
Author(s):  
Liang Shen ◽  
Yang Yang ◽  
Fei Ye ◽  
Gaoshan Liu ◽  
Marc Desforges ◽  
...  
Keyword(s):  
Reporter Gene ◽  
Luciferase Reporter ◽  
Wild Type Virus ◽  
Upper Respiratory Tract ◽  
Rna Helicases ◽  
Luciferase Reporter Gene ◽  
Screening Assay ◽  
Content Type ◽  
Tract Infections

ABSTRACTHuman coronaviruses (HCoVs) cause 15 to 30% of mild upper respiratory tract infections. However, no specific antiviral drugs are available to prevent or treat HCoV infections to date. Here, we developed four infectious recombinant HCoVs-OC43 (rHCoVs-OC43) which express theRenillaluciferase (Rluc) reporter gene. Among these four rHCoVs-OC43, rOC43-ns2DelRluc (generated by replacing ns2 with the Rluc gene) showed robust luciferase activity with only a slight impact on its growth characteristics. Additionally, this recombinant virus remained stable for at least 10 passages in BHK-21 cells. rOC43-ns2DelRluc was comparable to its parental wild-type virus (HCoV-OC43-WT) with respect to the quantity of the antiviral activity of chloroquine and ribavirin. We showed that chloroquine strongly inhibited HCoV-OC43 replicationin vitro, with a 50% inhibitory concentration (IC50) of 0.33 μM. However, ribavirin showed inhibition of HCoV-OC43 replication only at high concentrations which may not be applicable to humans in clinical treatment, with an IC50of 10 μM. Furthermore, using a luciferase-based small interfering RNA (siRNA) screening assay, we identified double-stranded-RNA-activated protein kinase (PKR) and DEAD box RNA helicases (DDX3X) that exhibited antiviral activities, which were further verified by the use of HCoV-OC43-WT. Therefore, rOC43-ns2DelRluc represents a promising safe and sensitive platform for high-throughput antiviral screening and quantitative analysis of viral replication.

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 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.

scholarly journals Cloning and Functional Analysis of Rat Tweety-Homolog 1 Gene Promoter

2021 ◽  
Author(s):  
Malgorzata Gorniak-Walas ◽  
Karolina Nizinska ◽  
Katarzyna Lukasiuk
Keyword(s):  
Transcriptional Regulation ◽  
Reporter Gene ◽  
Hippocampal Neurons ◽  
Gene Promoter ◽  
Promoter Sequence ◽  
Regulatory Elements ◽  
Luciferase Reporter ◽  
Luciferase Reporter Gene ◽  
Gene Assay

AbstractTweety-homolog 1 protein (Ttyh1) is abundantly expressed in neurons in the healthy brain, and its expression is induced under pathological conditions. In hippocampal neurons in vitro, Ttyh1 was implicated in the regulation of primary neuron morphology. However, the mechanisms that underlie transcriptional regulation of the Ttyh1 gene in neurons remain elusive. The present study sought to identify the promoter of the Ttyh1 gene and functionally characterize cis-regulatory elements that are potentially involved in the transcriptional regulation of Ttyh1 expression in rat dissociated hippocampal neurons in vitro. We cloned a 592 bp rat Ttyh1 promoter sequence and designed deletion constructs of the transcription factors specificity protein 1 (Sp1), E2F transcription factor 3 (E2f3), and achaete-scute homolog 1 (Ascl1) that were fused upstream of a luciferase reporter gene in pGL4.10[luc2]. The luciferase reporter gene assay showed the possible involvement of Ascl1, Sp1, and responsive cis-regulatory elements in Ttyh1 expression. These findings provide novel information about Ttyh1 gene regulation in neurons.

scholarly journals Novel MicroRNA Involved in Host Response to Avian Pathogenic Escherichia coli Identified by Deep Sequencing and Integration Analysis

2016 ◽  
Vol 85 (1) ◽  
Author(s):  
Xinzheng Jia ◽  
Qinghua Nie ◽  
Xiquan Zhang ◽  
Lisa K. Nolan ◽  
Susan J. Lamont
Keyword(s):  
Escherichia Coli ◽  
Host Resistance ◽  
Host Response ◽  
Differentially Expressed ◽  
Luciferase Reporter ◽  
Effective Control ◽  
Luciferase Reporter Gene ◽  
Content Type ◽  
Differentially Expressed Mirnas

ABSTRACT Avian pathogenic Escherichia coli (APEC) causes one of the most common bacterial diseases of poultry worldwide. Effective control methods are therefore desirable and will be facilitated by a better understanding of the host response to the pathogen. Currently, microRNAs (miRNAs) involved in host resistance to APEC are unknown. Here, we applied RNA sequencing to explore the changed miRNAs and deregulated genes in the spleen of three groups of broilers: nonchallenged (NC), APEC-challenged with mild pathology (CM), and APEC-challenged with severe pathology (CS). Twenty-seven differentially expressed miRNAs (fold change >1.5; P value <0.01) were identified, including 13 miRNAs between the NC and CM, 17 between the NC and CS, and 14 between the CM and CS groups. Through functional analysis of these miRNA targets, 12 immune-related biological processes were found to be significantly enriched. Based on combined analyses of differentially expressed miRNAs and mRNAs within each of the three groups, 43 miRNA-mRNA pairs displayed significantly negative correlations (r < −0.8). Notably, gga-miR-429 was greatly increased in the CS group compared to levels in both the CM and NC groups. In vitro, gga-miR-429 directly repressed luciferase reporter gene activity via binding to 3′ untranslated regions of TMEFF2, NTRK2, and SHISA2. Overexpression of gga-miR-429 in the HD11 macrophage cell line significantly inhibited TMEFF2 and SHISA2 expression, which are involved in the lipopolysaccharide-induced platelet-derived growth factor (PDGF) and Wnt signaling pathways. In summary, we provide the first report characterizing the miRNA changes during APEC infection, which may help to shed light on the roles of these recently identified genetic elements in the mechanisms of host resistance and susceptibility to APEC.

Direct Assessment of Promoter Activity of Human Cytochrome P450 Genes Using Optimized Transfection in Vitro and in Vivo

2006 ◽  
Vol 26 (3) ◽  
pp. 217-229 ◽  
Author(s):  
Mohammed Al-Dosari ◽  
Guisheng Zhang ◽  
Joseph E. Knapp ◽  
Dexi Liu
Keyword(s):  
Cytochrome P450 ◽  
Reporter Gene ◽  
Luciferase Reporter ◽  
Luciferase Expression ◽  
Promoter Strength ◽  
Luciferase Reporter Gene ◽  
Intact Mouse ◽  
Promoter Sequences ◽  
Human Cytochrome

Although transcription regulation of human cytochrome P450 enzymes (CYP) is known to play an important role in drug metabolism and homeostasis, factors influencing the expression of various CYP genes in humans remain largely undefined. We used three cell lines and CD-1 mice to assess the activity of genomic promoter sequences of human CYP2D6, 1A2, 3A4, 2C9, 2C18, and 2E1 genes. CYP promoter sequences were amplified by PCR using human liver genomic DNA as the template and cloned into pGL3-Basic vectors that contain a luciferase reporter gene but lack promoter or enhancer sequences. Each plasmid construct was transfected into cells in vitro using polyethylenimine (PEI) as the transfection reagent and into mice using the recently developed hydrodynamics-based procedure. Relative promoter strength was determined by the level of luciferase expression in transfected cells. All six human CYP promoters are active in driving reporter gene expression in cultured hepatic HepG2 cells and non-hepatic cells such as human embryonic kidney fibroblasts (293 cells) and murine melanoma cells (BL-16) as well as cells in intact mouse liver, lung, heart, kidney and spleen. The order of strength among CYP promoters examined was found to be 2D6 > 1A2 > 3A4 > 2C9 > 2C18 > 2E1.

scholarly journals Transcriptional Regulation of CYP3A4/2B6/2C9 Mediated via Nuclear Receptor PXR by Helicid and Its Metabolites

2015 ◽  
Vol 2015 ◽  
pp. 1-9
Author(s):  
Qun Chen ◽  
Hai-tang Xie ◽  
Yan Li ◽  
Guo Wang ◽  
Zhe Xu ◽  
...  
Keyword(s):  
Transcriptional Regulation ◽  
Nuclear Receptor ◽  
Reporter Gene ◽  
Negative Control Group ◽  
Negative Control ◽  
Luciferase Reporter ◽  
Control Group ◽  
Luciferase Reporter Gene ◽  
Expression Plasmid

Objective. This study aims at establishing and validating an in vitro system to screen drug inducers of CYPs mediated via hPXR, as well as studying transcriptional regulation of CYPs mediated via hPXR by helicid and its two metabolites.Methods. Cloning the nuclear receptor hPXR and the promoters of CYP3A4, CYP2B6, CYP2C9, and inserting the trans-element to the upstream of firefly luciferase reporter gene of the pGL4.17 vectors, then cotransfecting the report vectors and hPXR expression plasmid to HepG2 cell line. After 24 hours, the transfected cells were treated with helicid (0.004, 0.04, and 0.4 μmol/L) and its metabolite I and metabolite II (0.0004, 0.004, and 0.04 μmol/L) for 48 h, while rifampin (10 μmol/L) was included as the positive control and 0.1% DMSO as the negative control group. Cells were lysized and luciferase activity was determined using a dual luciferase reporter assay kit.Results. Helicid and its metabolites did not significantly increase promoter activities of CYP3A4, CYP2B6, and CYP2C9 in HepG2 cells transfected with PXR expression plasmid (P>0.05).Conclusion. PXR-expressed CYP3A4, CYP2B6, and CYP2C9 dual luciferase reporter gene platforms were successfully established, and helicid and its metabolites I, II do not significantly induce the transcription of CYP3A4, CYP2B6, and CYP2C9.

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.

scholarly journals LINC01213 promotes prostate cancer cell progression through miR-597/ BCL2L1 axis

2021 ◽  
Author(s):  
Xian Zhao ◽  
Xiaojing Xu ◽  
Qiong Wang ◽  
Xiaofei Wu
Keyword(s):  
Prostate Cancer ◽  
Reporter Gene ◽  
Reporter Gene Assay ◽  
Luciferase Reporter ◽  
Luciferase Reporter Gene ◽  
Western Blot Assay ◽  
Luciferase Reporter Gene Assay ◽  
Gene Assay

Abstract Background: Majority of cancer related deaths in males are attributed to prostate cancer (PRAD) throughout the world. Recently, the role of long non-coding RNAs (lncRNAs) in the pathogenesis of cancer has been widely explored. In this study, we investigated the role of lncRNA LINC01213 (LINC01213) in tumorigenesis of prostate cancer (PRAD).Methods: PRAD and adjacent tissue samples were collected from cancer patients. Survival rate among these patients was compared by Kaplan–Meier analysis. PRAD cells viability was estimated by CCK-8 method while AnnexinV/PI cytometry assay was used to determine the percent of apoptotic cells. qRT-PCR and western blot assay were used to determine the mRNA and protein expressions, respectively. Interaction between LINC01213 and corresponding miRNA as well as between miRNA and mRNA was confirmed by dual luciferase reporter gene assay. PRAD cells were also injected subcutaneously in nude mice to support in vitro findings.Results: It was observed that LINC01213 was highly expressed in PRAD samples and cell lines. Down-regulation of LINC01213 in PRAD cells decreased cell viability and inhibited proliferation. Luciferase reporter gene assay and RNA pull-down confirmed that LINC01213 targeted miR-597-3p. Increased expression of miR-597-3p resulted in decreased BCL2L2 expression in vitro. Inhibitory effects of miR-597-3p on PRAD cells’ survival and growth were diminished after LINC01213 overexpression which was also associated with alteration in the protein expression of BCL-xL, BCL-2 as well as caspase 3 and caspase 9.Conclusion: Taken together, our findings suggest that LINC01213 plays its role in PRAD tumorigenesis through miR-597-3p/ BCL2L2 dependent pathway with associated modulation of genes involved in cell survival and apoptosis.

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