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Reporter Expression (reporter + expression)
Selected AbstractsFunctional analysis of synaptotagmin gene regulatory regions in two distantly related ascidian speciesDEVELOPMENT GROWTH & DIFFERENTIATION, Issue 7 2008Jun Matsumoto We have studied the structure and function of a promoter region of the Halocynthia synaptotagmin (Hr-Syt) gene, which is abundantly expressed in neuronal cells. Our previous analysis suggested that the expression of Hr-Syt is regulated by at least one epidermal and two neuronal regulatory regions. In this study, the regulatory regions of Hr-Syt promoter were further characterized by using two species of ascidians, Halocynthia roretzi and Ciona intestinalis embryos. A putative GATA transcription factor binding site in the epidermal regulatory region has ectodermal enhancer activity in the Halocynthia embryo. Neuronal expression of Hr-Syt was regulated by multiple redundant enhancer regions. Among these enhancer regions, a 200-bp (,2900/,2700) region drove the reporter expression in neurons in both species of ascidian. Although the synaptotagmin promoter sequences did not show overall similarity between Hr-Syt and Ciona synaptotagmin (Ci-Syt), 5,-upsteream two short sequences of Ci-Syt have similarity to the ,2766/,2732 region of the Hr-Syt promoter. The homeodomain binding sites in this region are required for the neuronal enhancer activity. These results suggest that GATA and homeodomain transcription factors regulate the expression of synaptotagmin. [source] Enhancer detection in zebrafish permits the identification of neuronal subtypes that express Hox4 paralogsDEVELOPMENTAL DYNAMICS, Issue 8 2008Beena Punnamoottil Abstract Activity of zebrafish hoxb4a in the developing brain was analyzed in comparison to hoxa4a and hoxd4a using unique enhancer detection transgenes. Cytoplasmic YFP revealed shape and axonal projections of neurons in animals with insertions near the Hox4 genes and provided a means for the identification of neuronal subtypes. Despite an early activity of the genes in neuroepithelial cells and later in immature postmitotic neurons, we found reporter expression in distinct neuronal subtypes in the r7,r8-derived hindbrain. Most strikingly, hoxb4a neuronal subtypes projected through the vagus and into the pectoral fin while others formed symmetrically located fiber tracts innervating the cerebellum and the tectum, features that are partially shared by the other two paralogs. Collectively, our expression analysis indicates that hoxb4a in combination with its paralogs may play a significant role in the development of precerebellar, vagal, and pectoral fin neuronal subtypes. Developmental Dynamics 237:2195,2208, 2008. © 2008 Wiley-Liss, Inc. [source] Peripheral myelin protein 22 is regulated post-transcriptionally by miRNA-29a,GLIA, Issue 12 2009Jonathan D. Verrier Abstract Peripheral myelin protein 22 (PMP22) is a dose-sensitive, disease-associated protein primarily expressed in myelinating Schwann cells. Either reduction or overproduction of PMP22 can result in hereditary neuropathy, suggesting a requirement for correct protein expression for peripheral nerve biology. PMP22 is post-transcriptionally regulated and the 3,untranslated region (3,UTR) of the gene exerts a negative effect on translation. MicroRNAs (miRNAs) are small regulatory molecules that function at a post-transcriptional level by targeting the 3,UTR in a reverse complementary manner. We used cultured Schwann cells to demonstrate that alterations in the miRNA biogenesis pathway affect PMP22 levels, and endogenous PMP22 is subjected to miRNA regulation. GW-body formation, the proposed cytoplasmic site for miRNA-mediated repression, and Dicer expression, an RNase III family ribonuclease involved in miRNA biogenesis, are co-regulated with the differentiation state of Schwann cells. Furthermore, the levels of Dicer inversely correlate with PMP22, while the inhibition of Dicer leads to elevated PMP22. Microarray analysis of actively proliferating and differentiated Schwann cells, in conjunction with bioinformatics programs, identified several candidate PMP22-targeting miRNAs. Here we demonstrate that miR-29a binds and inhibits PMP22 reporter expression through a specific miRNA seed binding region. Over-expression of miR-29a enhances the association of PMP22 RNA with Argonaute 2, a protein involved in miRNA function, and reduces the steady-state levels of PMP22. In contrast, inhibition of endogenous miR-29a relieves the miRNA-mediated repression of PMP22. Correlation analyses of miR-29 and PMP22 in sciatic nerves reveal an inverse relationship, both developmentally and in post-crush injury. These results identify PMP22 as a target of miRNAs and suggest that myelin gene expression by Schwann cells is regulated by miRNAs. © 2009 Wiley-Liss, Inc. [source] Heregulin and forskolin-induced cyclin D3 expression in Schwann cells: Role of a CCAAT promoter element and CCAAT enhancer binding proteinGLIA, Issue 3 2004Luis Fuentealba Abstract Heregulin, a polypeptide growth factor, and forskolin, an adenylyl cyclase activator, synergistically stimulate expression of cyclin D3 and cell division in Schwann cells. Heregulin induces expression in Schwann cells of a luciferase reporter gene linked to the cyclin D3 promoter. Forskolin markedly augments reporter expression in the presence of heregulin. Deletion analysis identified several promoter sites that contribute to high-level reporter expression in heregulin- and forskolin-treated Schwann cells. A promoter fragment that contains 103 bp of 5,-flanking sequence produced significant reporter expression in heregulin- and forskolin-stimulated cells. Deletion of a consensus CCAAT site within this promoter fragment caused a nearly complete loss of reporter expression. Similar results were obtained when CCAAT site mutations were introduced into the promoter. Heregulin and forskolin increased steady-state levels of CCAAT/enhancer binding protein-, (C/EBP,) in Schwann cells. Mobility shift assays identified proteins in Schwann cell nuclear extracts that formed stable complexes with the cyclin D3 CCAAT promoter element and were disrupted by anti-C/EBP, antibody. Transfection of Schwann cells with C/EBP, cDNA increased cyclin D3 reporter expression. In contrast to these results, mutation of a cAMP response element in the cyclin D3 promoter had only a modest effect on heregulin- and forskolin-stimulated reporter expression. These findings demonstrate that C/EBP, plays a key role in the heregulin and cAMP-dependent regulation of cyclin D3 expression in Schwann cells. © 2003 Wiley-Liss, Inc. [source] Regulation of Dlx gene expression in the zebrafish pharyngeal arches: from conserved enhancer sequences to conserved activityJOURNAL OF APPLIED ICHTHYOLOGY, Issue 2 2010R. B. MacDonald Summary The Dlx genes play an important role in the development of the pharyngeal arches and the structures derived from these tissues, including the craniofacial skeleton. They are typically expressed in a nested pattern along the proximo-distal axis of the branchial arches in mice. This pattern is known as the "Dlx code" and has been proposed to be responsible for an early regional patterning of branchial arches in mammals. A number of cis - regulatory elements (CREs) have been identified within the Dlx loci, which target reporter expression to the developing pharyngeal arches of the mouse. Most of these CREs are located in the intergenic regions between the two genes constituting a Dlx bigene cluster. Therefore, the regionalized dlx expression in the branchial arches could be the result of the shared activities of these regulatory regions. Here we analyze previously published and new results showing these CREs are highly conserved in both their sequence and their activity in the pharyngeal arches of two distantly related vertebrates, the zebrafish and the mouse. A better understanding of Dlx gene regulation of the Dlx genes and of the genetic cascades in which they are involved can lead to clues explaining variations in morphology of the craniofacial regions of vertebrates. [source] Ndrg4 enhances NGF-induced ERK activation uncoupled with Elk-1 activationJOURNAL OF CELLULAR BIOCHEMISTRY, Issue 1 2006Shigeki Hongo Abstract Ndrg4 is expressed predominantly in the early postnatal rat brain and may be related to neural cell differentiation. PC12 cell lines stably expressing increased levels of Ndrg4 protein display enhanced NGF-induced phosphorylation of MEK and ERK. In contrast, the Ndrg4-C2-overexpressed PC12 cell lines showed attenuated NGF-promoted phosphorylation of Elk-1, which is a nuclear target of ERK. A reporter assay also indicated that Ndrg4-C2 suppresses Elk-1-mediated transcriptional activation and SRE reporter expression. The suppressive effect of Ndrg4-C2 on NGF-induced activation of Elk-1 was abolished by colchicine but not by cytochalasin D, suggesting that microtubules are involved in the reduced activation of Elk-1 by Ndrg4. Ndrg4 may play a role in supporting the activation of ERK and its target proteins needed for neuronal differentiation and in reducing the activation of Elk-1 implicated in cell growth. J. Cell. Biochem. 98: 185,193, 2006. © 2006 Wiley-Liss, Inc. [source] HER2 signaling enhances 5,UTR-mediated translation of c-Myc mRNAJOURNAL OF CELLULAR PHYSIOLOGY, Issue 1 2004Enrico Galmozzi The increased levels of c-Myc protein observed previously in an ovarian carcinoma cell line stably transfected to express HER2 has suggested a role for the HER2 pathway in c-Myc expression. Analysis of HER2-transfected cells stimulated with heregulin ,1 (HRG) revealed increased c-Myc protein levels but not a corresponding increase in c-Myc mRNA expression or any change in c-Myc protein half-life. Transfection of HER2-overexpressing cells with a construct containing the 5, untranslated region (5,UTR) of c-Myc mRNA originated from the P2 promoter and placed upstream of the Renilla luciferase gene, enhanced reporter expression upon stimulation with HRG. The HRG-mediated increase in reporter activity correlated with the HRG-mediated induction observed for c-Myc protein, identifying the P2-derived leader (P2L) of c-Myc mRNA as the cis -element involved in c-Myc translational induction. Both the increase in c-Myc protein levels and P2L-enhanced translational activity were inhibited by the PI3K inhibitor wortmannin. Together, these results demonstrate that HRG stimulation of HER2 overexpressing cells leads to enhanced c-Myc protein synthesis through activation of the PI3K/Akt/mTOR pathway and that the P2L of c-Myc mRNA is the element responsible for induction of c-Myc translation. © 2004 Wiley-Liss, Inc. [source] Lentivirus-mediated bifunctional cell labeling for in vivo melanoma studyPIGMENT CELL & MELANOMA RESEARCH, Issue 3 2009Chi-Ping Day Summary Lentiviral vectors (LVs) are capable of labeling a broad spectrum of cell types, achieving stable expression of transgenes. However, for in vivo studies, the duration of marker gene expression has been highly variable. We have developed a series of LVs harboring different promoters for expressing reporter gene in mouse cells. Long-term culture and colony formation of several LV-labeled mouse melanoma cells showed that promoters derived from mammalian house-keeping genes, especially those encoding RNA polymerase II (Pol2) and ferritin (FerH), provided the highest consistency for reporter expression. For in vivo studies, primary B16BL6 mouse melanoma were infected with LVs whose luciferase,green fluorescence protein fusion gene (Luc/GFP) was driven by either Pol2 or FerH promoters. When transplanted into syngeneic C57BL/6 mice, Luc/GFP-labeled B16BL6 mouse melanoma cells can be monitored by bioluminescence imaging in vivo, and GFP-positive cells can be isolated from the tumors by fluorescence-activated cell sorter. Pol2-Luc/GFP labeling, while lower in activity, was more sustainable than FerH-Luc/GFP labeling in B16BL6 over consecutive passages into mice. We conclude that Pol-2-Luc/GFP labeling allows long-term in vivo monitoring and tumor cell isolation in immunocompetent mouse melanoma models. [source] Ethylene regulates lateral root formation and auxin transport in Arabidopsis thalianaTHE PLANT JOURNAL, Issue 2 2008Sangeeta Negi Summary Lateral root branching is a genetically defined and environmentally regulated process. Auxin is required for lateral root formation, and mutants that are altered in auxin synthesis, transport or signaling often have lateral root defects. Crosstalk between auxin and ethylene in root elongation has been demonstrated, but interactions between these hormones in the regulation of Arabidopsis lateral root formation are not well characterized. This study utilized Arabidopsis mutants altered in ethylene signaling and synthesis to explore the role of ethylene in lateral root formation. We find that enhanced ethylene synthesis or signaling, through the eto1-1 and ctr1-1 mutations, or through the application of 1-aminocyclopropane-1-carboxylic acid (ACC), negatively impacts lateral root formation, and is reversible by treatment with the ethylene antagonist, silver nitrate. In contrast, mutations that block ethylene responses, etr1-3 and ein2-5, enhance root formation and render it insensitive to the effect of ACC, even though these mutants have reduced root elongation at high ACC doses. ACC treatments or the eto1-1 mutation significantly enhance radiolabeled indole-3-acetic acid (IAA) transport in both the acropetal and the basipetal directions. ein2-5 and etr1-3 have less acropetal IAA transport, and transport is no longer regulated by ACC. DR5-GUS reporter expression is also altered by ACC treatment, which is consistent with transport differences. The aux1-7 mutant, which has a defect in an IAA influx protein, is insensitive to the ethylene inhibition of root formation. aux1-7 also has ACC-insensitive acropetal and basipetal IAA transport, as well as altered DR5-GUS expression, which is consistent with ethylene altering AUX1-mediated IAA uptake, and thereby blocking lateral root formation. [source] Inhibition of cartilage degradation: A combined tissue engineering and gene therapy approachARTHRITIS & RHEUMATISM, Issue 3 2003Wael Kafienah Objective To determine if tissue-engineered cartilage can be protected from cytokine-induced degradation using a gene therapy approach. Methods Chemical and pantropic retroviral gene transfer methodologies were compared for their ability to introduce a luciferase reporter gene into adult bovine cartilage chondrocytes grown in monolayer. Pantropic retrovirus was then used to transduce these cells with human tissue inhibitor of metalloproteinases 1 (TIMP-1), and the stability of expression in monolayer or pellet culture was monitored for 6 weeks. Untransduced and TIMP-1,transduced cells were also used to tissue engineer 3-dimensional cartilage constructs that were then challenged with interleukin-1 (IL-1) for 4 weeks. Conditioned media and residual cartilage were collected for analysis of matrix components, including type II collagen and proteoglycans, and for TIMP-1 production and matrix metalloproteinase (MMP) activity. Results Chemical transfection of adult bovine chondrocytes gave rise to short-lived reporter expression that was virtually undetectable after 4 weeks of culture. In contrast, pantropic retroviral transduction gave rise to stable expression that persisted at a high level for at least 6 weeks. Pantropic transduction of the cells with TIMP-1 gave rise to similar long-term expression, both in monolayer and pellet cultures. TIMP-1,transduced tissue-engineered cartilage also retained TIMP-1 expression for an additional 4 weeks of culture in the presence of IL-1. Compared with control samples, TIMP-1,transgenic cartilage resisted the catabolic effects of IL-1, with MMP activity reduced to basal levels and a decreased loss of type II collagen. Conclusion Pantropic retroviral transduction permits long-term expression of potentially therapeutic transgenes in adult tissue-engineered cartilage. While TIMP-1 transduction could be used to prevent collagen breakdown, alternative transgenes may be necessary to protect cartilage proteoglycans. [source] Continuous wave and simulated GSM exposure at 1.8 W/kg and 1.8 GHz do not induce hsp16-1 heat-shock gene expression in Caenorhabditis elegansBIOELECTROMAGNETICS, Issue 2 2008Adam S. Dawe Abstract Recent data suggest that there might be a subtle thermal explanation for the apparent induction by radiofrequency (RF) radiation of transgene expression from a small heat-shock protein (hsp16-1) promoter in the nematode, Caenorhabditis elegans. The RF fields used in the C. elegans study were much weaker (SAR 5,40 mW,kg,1) than those routinely tested in many other published studies (SAR ,2 W,kg,1). To resolve this disparity, we have exposed the same transgenic hsp16-1::lacZ strain of C. elegans (PC72) to higher intensity RF fields (1.8 GHz; SAR ,1.8 W,kg,1). For both continuous wave (CW) and Talk-pulsed RF exposures (2.5 h at 25 °C), there was no indication that RF exposure could induce reporter expression above sham control levels. Thus, at much higher induced RF field strength (close to the maximum permitted exposure from a mobile telephone handset), this particular nematode heat-shock gene is not up-regulated. However, under conditions where background reporter expression was moderately elevated in the sham controls (perhaps as a result of some unknown co-stressor), we found some evidence that reporter expression may be reduced by ,15% following exposure to either Talk-pulsed or CW RF fields. Bioelectromagnetics 29:92,99, 2008. © 2007 Wiley-Liss, Inc. [source] A small temperature rise may contribute towards the apparent induction by microwaves of heat-shock gene expression in the nematode Caenorhabditis ElegansBIOELECTROMAGNETICS, Issue 2 2006Adam S. Dawe Abstract We have previously reported that low intensity microwave exposure (0.75,1.0 GHz CW at 0.5 W; SAR 4,40 mW/kg) can induce an apparently non-thermal heat-shock response in Caenorhabditis elegans worms carrying hsp16-1::reporter genes. Using matched copper TEM cells for both sham and exposed groups, we can detect only modest reporter induction in the latter exposed group (15,20% after 2.5 h at 26 °C, rising to ,50% after 20 h). Traceable calibration of our copper TEM cell by the National Physical Laboratory (NPL) reveals significant power loss within the cell (8.5% at 1.0 GHz), accompanied by slight heating of exposed samples (,0.3 °C at 1.0 W). Thus, exposed samples are in fact slightly warmer (by ,0.2 °C at 0.5 W) than sham controls. Following NPL recommendations, our TEM cell design was modified with the aim of reducing both power loss and consequent heating. In the modified silver-plated cell, power loss is only 1.5% at 1.0 GHz, and sample warming is reduced to ,0.15 °C at 1.0 W (i.e., ,0.1 °C at 0.5 W). Under sham:sham conditions, there is no difference in reporter expression between the modified silver-plated TEM cell and an unmodified copper cell. However, worms exposed to microwaves (1.0 GHz and 0.5 W) in the silver-plated cell also show no detectable induction of reporter expression relative to sham controls in the copper cell. Thus, the 20% "microwave induction" observed using two copper cells may be caused by a small temperature difference between sham and exposed conditions. In worms incubated for 2.5 h at 26.0, 26.2, and 27.0 °C with no microwave field, there is a consistent and significant increase in reporter expression between 26.0 and 26.2 °C (by ,20% in each of the six independent runs), but paradoxically expression levels at 27.0 °C are similar to those seen at 26.0 °C. This surprising result is in line with other evidence pointing towards complex regulation of hsp16-1 gene expression across the sub-heat-shock range of 25,27.5 °C in C. elegans. We conclude that our original interpretation of a non-thermal effect of microwaves cannot be sustained; at least part of the explanation appears to be thermal. Bioelectromagnetics 27:88,97, 2006. © 2005 Wiley-Liss, Inc. [source] | ||||||||||