scholarly journals Identification and characterization of a silencer regulatory element in the 3′-flanking region of the murine CD46 gene

2000 ◽  
Vol 351 (2) ◽  
pp. 353-365 ◽  
Author(s):  
Midori NOMURA ◽  
Akira TSUJIMURA ◽  
Nasim A. BEGUM ◽  
Misako MATSUMOTO ◽  
Hiroetsu WABIKO ◽  
...  
Keyword(s):  
Gene Expression ◽  
Reporter Gene ◽  
Stop Codon ◽  
Regulatory Element ◽  
Luciferase Reporter ◽  
Size Difference ◽  
Binding Factor ◽  
Gene Assay ◽  
Flanking Region ◽  
Silencer Element

The murine membrane cofactor protein (CD46) gene is expressed exclusively in testis, in contrast to human CD46, which is expressed ubiquitously. To elucidate the mechanism of differential CD46 gene expression among species, we cloned entire murine CD46 genomic DNA and possible regulatory regions were placed in the flanking region of the luciferase reporter gene. The reporter gene assay revealed a silencing activity not in the promoter, but in the 3´-flanking region of the gene and the silencer-like element was identified within a 0.2-kb region between 0.6 and 0.8kb downstream of the stop codon. This silencer-like element was highly similar to that of the pig MHC class-I gene. The introduction of a mutation into this putative silencer element of murine CD46 resulted in an abrogation of the silencing effect. Electrophoretic mobility-shift assay indicated the presence of the binding molecule(s) for this silencer sequence in murine cell lines and tissues. A size difference of the protein–silencer-element complex was observed depending upon the solubilizers used for preparation of the nuclear extracts. A mutated silencer sequence failed to interact with the binding molecules. The level of the binding factor was lower in the testicular germ cells compared with other organs. Thus the silencer element and its binding factor may play a role in transcriptional regulation of murine CD46 gene expression. These results imply that the effects of the CD46 silencer element encompass the innate immune and reproductive systems, and in mice may determine the testicular germ-cell-dominant expression of CD46.

scholarly journals Creating a human-induced pluripotent stem cell-based NKX2.5 reporter gene assay for developmental toxicity testing

2021 ◽  
Author(s):  
Karin Lauschke ◽  
Andreas Frederik Treschow ◽  
Mikkel Aabech Rasmussen ◽  
Nichlas Davidsen ◽  
Bjørn Holst ◽  
...  
Keyword(s):  
Stem Cell ◽  
Reporter Gene ◽  
Luminescence Intensity ◽  
Developmental Toxicity ◽  
Embryonic Stem ◽  
Embryoid Bodies ◽  
Luciferase Reporter ◽  
Reporter Cell ◽  
Gene Assay ◽  
Induced Pluripotent

AbstractTo test large numbers of chemicals for developmental toxicity, rapid in vitro tests with standardized readouts for automated data acquisition are needed. However, the most widely used assay, the embryonic stem cell test, relies on the counting of beating embryoid bodies by visual inspection, which is laborious and time consuming. We previously developed the PluriBeat assay based on differentiation of human induced pluripotent stem cells (hiPSC) that we demonstrated to be predictive for known teratogens at relevant concentrations using the readout of beating cardiomyocytes. Here, we report the development of a novel assay, which we term the PluriLum assay, where we have introduced a luciferase reporter gene into the locus ofNKX2.5of our hiPSC line. This enabled us to measure luminescence intensities instead of counting beating cardiomyocytes, which is less labor intensive. We established twoNKX2.5reporter cell lines and validated their pluripotency and genetic stability. Moreover, we confirmed that the genetically engineeredNKX2.5reporter cell line differentiated into cardiomyocytes with the same efficiency as the original wild-type line. We then exposed the cells to valproic acid (25–300 μM) and thalidomide (0.1–36 µM) and compared the PluriBeat readout of the cardiomyocytes with the luminescence intensity of the PluriLum assay. The results showed that thalidomide decreased luminescence intensity significantly with a higher potency and efficacy compared to the beating readout. With this, we have developed a novel hiPSC-based assay with a standardized readout that may have the potential for higher throughput screening for developmental toxicity.

Abstract 117: MicroRNA-138 regulates S100A1 in Endothelial Cells

2013 ◽  
Vol 113 (suppl_1) ◽  
Author(s):  
Anagha Sen ◽  
Shumei Ren ◽  
Jianxin Sun ◽  
Patrick Most ◽  
Karsten Peppel
Keyword(s):  
Gene Expression ◽  
Endothelial Cells ◽  
Reporter Gene ◽  
Recombinant Adenovirus ◽  
Limb Ischemia ◽  
Bioinformatic Analysis ◽  
Reporter Gene Expression ◽  
Luciferase Reporter ◽  
C2c12 Myotubes ◽  
Luciferase Reporter Construct

Rationale: The EF-hand Ca2+ sensor S100A1 is essential for proper endothelial nitric oxide (NO) synthase (eNOS) activation. S100A1 levels are greatly reduced in endothelial cells (ECs) subjected to hypoxia, rendering them dysfunctional. Objective: To determine if the 3’UTR mediates the rapid hypoxia-induced downregulation of S100A1 in ECs. Methods and Results: ECs transfected with a S100A1 - 3’ untranslated region (UTR) luciferase reporter construct displayed significantly reduced gene expression when subjected to gas or chemical hypoxia. Bioinformatic analysis suggested that microRNA -138 (miR-138) could target the 3’UTR of S100A1. Hypoxia greatly increased miR-138 levels in ECs, but not in skeletal muscle C2C12 myotubes. Consistent with this finding, patients with critical limb ischemia (CLI) or mice subjected to femoral artery resection (FAR) displayed increased miR-138 levels. Transfection of a miR-138 mimic into ECs reduced S100A1 - 3 ‘UTR reporter gene expression, while transfection of an anti miR-138 (antagomir) prevented the hypoxia-induced downregulation of the reporter gene. The increased levels of miR-138 are dependent on Hif1-α activation as treatment with siRNA against Hif1-α prevented S100A1 reporter gene downregulation after hypoxia. Conversely, specific activation of Hif1-α by a selective prolyl-hydroxylase inhibitor (IOX2) reduced reporter gene expression. Finally, ECs transfected with miR-138 mimic displayed reduced tube formation when plated onto Matrigel matrix and expressed less NO when stimulated with VEGF. These effects were reversed by gene transfer of S100A1 using recombinant adenovirus. Conclusions: Our study shows that miR-138 is an essential mediator of EC dysfunction via its ability to target the 3’UTR of S100A1 in a hypoxia-induced manner. MiR-138 might thus be an attractive target for the treatment of pathologies that are linked to endothelial dysfunction.

scholarly journals Sequence of the 5′-flanking region and promoter activity of the human mucin gene MUC5B in different phenotypes of colon cancer cells

2000 ◽  
Vol 348 (3) ◽  
pp. 675-686 ◽  
Author(s):  
Isabelle VAN SEUNINGEN ◽  
Michaël PERRAIS ◽  
Pascal PIGNY ◽  
Nicole PORCHET ◽  
Jean-Pierre AUBERT
Keyword(s):  
Gene Expression ◽  
Transcription Start Site ◽  
Promoter Activity ◽  
Luciferase Reporter ◽  
Nuclear Factor ◽  
Start Site ◽  
Transcription Start ◽  
Mucin Gene ◽  
Intron 1 ◽  
Flanking Region

Control of gene expression in intestinal cells is poorly understood. Molecular mechanisms that regulate transcription of cellular genes are the foundation for understanding developmental and differentiation events. Mucin gene expression has been shown to be altered in many intestinal diseases and especially cancers of the gastrointestinal tract. Towards understanding the transcriptional regulation of a member of the 11p15.5 human mucin gene cluster, we have characterized 3.55 kb of the 5ʹ-flanking region of the human mucin gene MUC5B, including the promoter, the first two exons and the first intron. We report here the promoter activity of successively 5ʹ-truncated sections of 956 bases of this region by fusing it to the coding region of a luciferase reporter gene. The transcription start site was determined by primer-extension analysis. The region upstream of the transcription start site is characterized by the presence of a TATA box at bases -32/-26, DNA-binding elements for transcription factors c-Myc, N-Myc, Sp1 and nuclear factor ĸB as well as putative activator protein (AP)-1-, cAMP-response-element-binding protein (CREB)-, hepatocyte nuclear factor (HNF)-1-, HNF-3-, TGT3-, gut-enriched Krüppel factor (GKLF)-, thyroid transcription factor (TTF)-1- and glucocorticoid receptor element (GRE)-binding sites. Intron 1 of MUC5B was also characterized, it is 2511 nucleotides long and contains a DNA segment of 259 bp in which are clustered eight tandemly repeated GA boxes and a CACCC box that bind Sp1. AP-2α and GATA-1 nuclear factors were also shown to bind to their respective cognate elements in intron 1. In transfection studies the MUC5B promoter showed a cell-specific activity as it is very active in mucus-secreting LS174T cells, whereas it is inactive in Caco-2 enterocytes and HT-29 STD (standard) undifferentiated cells. Within the promoter, maximal transcription activity was found in a segment covering the first 223 bp upstream of the transcription start site. Finally, in co-transfection experiments a transactivating effect of Sp1 on to MUC5B promoter was seen in LS174T and Caco-2 cells.

Potency of isothiocyanates to induce luciferase reporter gene expression via the electrophile-responsive element from murine glutathione S-transferase Ya

2009 ◽  
Vol 23 (4) ◽  
pp. 617-621 ◽  
Author(s):  
Martijn Vermeulen ◽  
Anne-Marie M.J.F. Boerboom ◽  
Barry M.G. Blankvoort ◽  
Jac M.M.J.G. Aarts ◽  
Ivonne M.C.M. Rietjens ◽  
...  
Keyword(s):  
Gene Expression ◽  
Reporter Gene ◽  
Reporter Gene Expression ◽  
Luciferase Reporter ◽  
Glutathione S Transferase ◽  
Luciferase Reporter Gene ◽  
Responsive Element

scholarly journals Response of the 5′-flanking region of the human 25-hydroxyvitamin D 1α-hydroxylase gene to physiological stimuli using a transgenic mouse model

2005 ◽  
Vol 34 (1) ◽  
pp. 237-245 ◽  
Author(s):  
I Hendrix ◽  
P H Anderson ◽  
J L Omdahl ◽  
B K May ◽  
H A Morris
Keyword(s):  
Gene Expression ◽  
Vitamin D ◽  
Vitamin D Deficiency ◽  
Transgenic Mouse ◽  
Reporter Gene ◽  
Dietary Calcium ◽  
Mrna Levels ◽  
Hydroxyvitamin D ◽  
25 Hydroxyvitamin D ◽  
Flanking Region

The enzyme 25-hydroxyvitamin D 1α-hydroxylase, or CYP27B1, is the key enzyme in the two-step activation process of vitamin D to 1,25-dihydroxyvitamin D (1,25D). While a number of regulators of the renal CYP27B1 enzyme activity have been recognized for some years, their underlying molecular mechanisms remain largely unknown, and the DNA regions involved in the in vivo regulation of gene expression by these factors have not been delineated. We have generated a transgenic mouse line that expresses 1501 bp of 5′ flanking region together with 44 bp of 5′ untranslated region of the human CYP27B1 gene fused to the firefly luciferase reporter gene. Animals expressing the luciferase gene demonstrated that both luciferase protein and mRNA for CYP27B1 were localized to proximal convoluted tubule cells of the kidney. In 2-week-old animals, the expression of the transgene and the endogenous CYP27B1 mRNA levels in the kidney were highest and fell with increasing age. Both reporter gene expression and CYP27B1 mRNA levels were downregulated in response to increasing amounts of dietary calcium in a dose-dependent manner. Vitamin D deficiency resulted in an increase in both the reporter gene and CYP27B1 expression. Interestingly, the increase in CYP27B1 mRNA levels was substantially higher than the increase in reporter gene expression, suggesting either that there is a post-transcriptional mechanism that increases the amount of CYP27B1 mRNA or that other regulatory elements are required to maximize the effect of vitamin D deficiency. These findings demonstrate that the 1501 bp 5′ flanking region of the CYP27B1 gene directs expression to the proximal convoluted tubules of the kidney and is responsible for increasing transcriptional activity when dietary calcium and vitamin D levels are depleted. It also responds in the kidney to the physiological regulators of development and ageing.

scholarly journals Non-invasive somatotransgenic bioimaging in living animals

F1000Research ◽  
2020 ◽  
Vol 9 ◽  
pp. 1216 ◽  
Author(s):  
Juliette M. Delhove ◽  
Rajvinder Karda ◽  
Lorna M. FitzPatrick ◽  
Suzanne M.K. Buckley ◽  
Simon N. Waddington ◽  
...  
Keyword(s):  
Gene Expression ◽  
Reporter Gene ◽  
Viral Vectors ◽  
Es Cells ◽  
Embryonic Stem ◽  
Whole Body ◽  
Luciferase Reporter ◽  
Gene Promoters ◽  
Luciferase Reporter Gene ◽  
Endogenous Gene

Bioluminescence imaging enables noninvasive quantification of luciferase reporter gene expression in transgenic tissues of living rodents. Luciferase transgene expression can be regulated by endogenous gene promoters after targeted knock-in of the reporter gene, usually within the first intron of the gene. Even using CRISPR/Cas9 mediated genome editing this can be a time consuming and costly process. The generation of germline transgenic (GLT) rodents by targeted genomic integration of a gene expression cassette in embryonic stem (ES) cells is commonplace but results in the wastage of large numbers of animals during colony generation, back-crossing and maintenance. Using a synthetic/truncated promoter-driven luciferase gene to study promoter activity in a given tissue or organ of a GLT also often results in unwanted background luciferase activity during whole-body bioluminescent imaging as every cell contains the reporter. We have developed somatotransgenic bioimaging; a method to generate tissue-restricted transcription factor activated luciferase reporter (TFAR) cassettes in rodents that substantially reduces the number of animals required for experimentation. Bespoke designed TFARs are delivered to newborn pups using viral vectors targeted to specific organs by tissue-tropic pseudotypes. Retention and proliferation of TFARs is facilitated by stem/progenitor cell transduction and immune tolerance to luciferase due to the naïve neonatal immune system. We have successfully applied both lentiviral and adeno-associated virus (AAV) vectors in longitudinal rodent studies, targeting TFARs to the liver and brain during normal development and in well-established disease models. Development of somatotransgenic animals has broad applicability to non-invasively determine mechanistic insights into homeostatic and disease states and assess toxicology and efficacy testing. Somatotransgenic bioimaging technology is superior to current whole-body, light-emitting transgenic models as it reduces the numbers of animals used by generating only the required number of animals. It is also a refinement over current technologies given the ability to use conscious, unrestrained animals.

In vivo regulation of β-MHC gene in rodent heart: role of T3 and evidence for an upstream enhancer

1999 ◽  
Vol 276 (4) ◽  
pp. C883-C891 ◽  
Author(s):  
Carola E. Wright ◽  
F. Haddad ◽  
A. X. Qin ◽  
P. W. Bodell ◽  
K. M. Baldwin
Keyword(s):  
Gene Expression ◽  
Thyroid Hormone ◽  
Reporter Gene ◽  
Normal Control ◽  
Transcriptional Activity ◽  
Luciferase Reporter ◽  
Luciferase Reporter Gene ◽  
Promoter Construct ◽  
Hormone Responsiveness

Cardiac β-myosin heavy chain (β-MHC) gene expression is mainly regulated through transcriptional processes. Although these results are based primarily on in vitro cell culture models, relatively little information is available concerning the interaction of key regulatory factors thought to modulate MHC expression in the intact rodent heart. Using a direct gene transfer approach, we studied the in vivo transcriptional activity of different-length β-MHC promoter fragments in normal control and in altered thyroid states. The test β-MHC promoter was fused to a firefly luciferase reporter gene, whereas the control α-MHC promoter was fused to the Renilla luciferase reporter gene and was used to account for variations in transfection efficiency. Absolute reporter gene activities showed that β- and α-MHC genes were individually and reciprocally regulated by thyroid hormone. The β-to-α ratios of reporter gene expression demonstrated an almost threefold larger β-MHC gene expression in the longest than in the shorter promoter fragments in normal control animals, implying the existence of an upstream enhancer. A mutation in the putative thyroid response element of the −408-bp β-MHC promoter construct caused transcriptional activity to drop to null. When studied in the −3,500-bp β-MHC promoter, construct activity was reduced (∼100-fold) while thyroid hormone responsiveness was retained. These findings suggest that, even though the bulk of the thyroid hormone responsiveness of the gene is contained within the first 215 bp of the β-MHC promoter sequence, the exact mechanism of triiodothyronine (T3) action remains to be elucidated.

scholarly journals Identification of Alkaloids from Corydalis yanhusuo W. T. Wang as Dopamine D1 Receptor Antagonists by Using CRE-Luciferase Reporter Gene Assay

Molecules ◽  
2018 ◽  
Vol 23 (10) ◽  
pp. 2585 ◽  
Author(s):  
Lehao Wu ◽  
Weiyue Zhang ◽  
Xin Qiu ◽  
Chaoran Wang ◽  
Yanfang Liu ◽  
...  
Keyword(s):  
Dopamine Receptors ◽  
Reporter Gene ◽  
Reporter Gene Assay ◽  
D1 Receptor ◽  
Assay System ◽  
Luciferase Reporter ◽  
Luciferase Reporter Gene ◽  
Luciferase Reporter Gene Assay ◽  
Antagonistic Effects ◽  
Gene Assay

Corydalis yanhusuo W. T. Wang (C. yanhusuo) has been traditionally used for drug addiction and pain relief in China. In our previous study, we showed that the extract of C. yanhusuo blocks dopamine receptors, demonstrating that its pharmacological activities are mostly due to the antagonistic effects of some of its components at dopamine receptors. As part of our ongoing project on C. yanhusuo, the aim of the present study is to establish a high-throughput and low-cost screening assay system and test the abilities of the isolated alkaloids from C. yanhusuo to inhibit dopamine-induced dopamine D1 receptor activity. By using our established cyclic adenosine monophosphate (cAMP)-response element (CRE)-luciferase reporter gene assay system, we identified eight alkaloids from C. yanhusuo with D1 receptor antagonistic activities. We next validated the activities of these compounds using fluorometric imaging plate reader (FLIPR) assay by measuring the intracellular Ca2+ change. Six out of eight compounds, including tetrahydropalmatine, corydaline, 13-methyldehydrocorydalmine, dehydrocorybubine, dehydrocorydaline, and columbamine, can be confirmed for their inhibitory activities. The dopamine-receptor-antagonistic effects of four compounds, including 13-methyldehydrocorydalmine, dehydrocorydaline, columbamine, and corydaline, are reported for the first time. The present study provides an important pharmacological basis to support the traditional use of C. yanhusuo in China.

scholarly journals eNOS Promoter Activation by Red Wine Polyphenols: an Interaction Study

2013 ◽  
Vol 60 (1) ◽  
pp. 27-33 ◽  
Author(s):  
E. Kurin ◽  
N. Fakhrudin ◽  
M. Nagy
Keyword(s):  
Reporter Gene ◽  
Red Wine ◽  
Interaction Analysis ◽  
Mass Action ◽  
Luciferase Reporter ◽  
Interaction Study ◽  
Luciferase Reporter Gene ◽  
Gene Assay ◽  
Wine Polyphenols ◽  
Red Wine Polyphenols

Beneficial effects of red wine polyphenols on cardiovascular health are well known. The aim of our research was an interaction study of four red wine polyphenols – resveratrol (R), quercetin (Q), kaempferol (KF) and isorhamnetin (IR) of their ability to activate endothelial NO synthase (eNOS) promoter when used alone and in equimolar mixtures. To determine their activity, we performed a luciferase reporter gene assay on EA.hy926 cells stably transfected with a luciferase reporter gene construct containing eNOS promoter. The Bradford assay was also performed to account the cytotoxicity and/or the cell number differences. The median effect equation, as an interaction analysis evaluating synergy or antagonism of the combinations was done according to mass-action law principle. Isobolographic method was performed on selected double mixtures and dose reduction index was calculated for all mixtures. All single polyphenols activated eNOS promoter. The EC50 values were in micromolar concentrations ranging from 3.44 μM (R2 = 0.96) for kaempferol to 9.89 μM for isorhamnetin (R2 = 0.94). All mixtures activated eNOS promoter, but their interactions varied from synergy (Q+R, Q+IR+KF, Q+R+KF and Q+R+IR+KF), through additive (R+IR+KF) to antagonistic interaction (R+IR, R+KF, Q+IR, Q+KF, IR+KF and R+Q+IR). In this study, we show for the first time that red wine polyphenols activated eNOS promoter when used alone and in mixtures with different type of interactions.

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