Search results for "Reporter"
showing 10 items of 166 documents
Desmosomes: interconnected calcium-dependent structures of remarkable stability with significant integral membrane protein turnover
2002
Desmosomes are prominent cell adhesion structures that are major stabilizing elements, together with the attached cytoskeletal intermediate filament network, of the cytokeratin type in epithelial tissues. To examine desmosome dynamics in tightly coupled cells and in situations of decreased adhesion, fluorescent desmosomal cadherin desmocollin 2a (Dsc2a) chimeras were stably expressed in human hepatocellular carcinoma-derived PLC cells (clone PDc-13) and in Madin-Darby canine kidney cells (clone MDc-2) for the continuous monitoring of desmosomes in living cells. The hybrid polypeptides integrated specifically and without disturbance into normal-appearing desmosomes that occurred in associati…
Transcription of human neuronal nitric oxide synthase mRNAs derived from different first exons is partly controlled by exon 1-specific promoter seque…
2006
AbstractThe human neuronal nitric oxide synthase (NOS1) gene is subject to extensive splicing. A total of 12 NOS1 mRNA species have been identified. They differ in their 5′ ends and are derived from 12 different first exons (termed exons 1a to 1l). Various cell lines whose NOS1 first exon expression patterns were representative of human brain, skin, and skeletal muscle were identified. These included A673 neuroepithelioma cells, SK-N-MC neuroblastoma cells, HaCaT keratinocyte-like cells, and C2C12 myocyte-like cells. In these cell lines, correlations were found between the exon 1 variants preferentially expressed and the promoter activities of their cognate 5′ flanking sequences. These data…
Transcriptional targeting of dendritic cells for gene therapy using the promoter of the cytoskeletal protein fascin.
2003
Strong cell-type-specific promoters are basic tools in gene therapy allowing for novel applications and focused strategies by transcriptionally targeting gene expression to selected cells. In immunotherapy, dendritic cells (DC) are of central importance, since they represent the principal inducers of immune responses. Here we describe isolation and use of the promoter of the murine actin-bundling protein fascin to target transcriptionally gene expression to cutaneous DC. Using the reporter gene enhanced green fluorescent protein (EGFP), we demonstrate that the fascin promoter mediates a strong antigen expression that is restricted to mature DC. DNA vaccination with antigen-encoding expressi…
Transcriptional targeting of dendritic cells in gene gun-mediated DNA immunization favors the induction of type 1 immune responses
2003
Cutaneous dendritic cells (DC) are pivotal for the elicitation of antigen-specific immune responses following gene gun-mediated biolistic transfection of the skin. We transcriptionally targeted transgene expression to DC using vectors containing the murine fascin promoter (pFascin) to control antigen production and compared the immune response elicited with conventional DNA immunization using plasmid constructs with the ubiquitously active CMV promoter (pCMV). Biolistic transfection with pFascin initiated a marked type 1 immune response characterized by the occurrence of a large population of IFN-gamma-producing T helper (Th) cells in spleen and draining lymph nodes. Consistently, immunoglo…
A major cysteine proteinase, EPB, in germinating barley seeds: structure of two intronless genes and regulation of expression
1996
The barley cysteine proteinase B (EPB) is the main protease responsible for the degradation of endosperm storage proteins providing nitrogenous nutrients to support the growth of young seedlings. The expression of this enzyme is induced in the germinating seeds by the phytohormone, gibberellin, and suppressed by another phytohormone, abscisic acid. In situ hybridization experiments indicate that EPB is expressed in the scutellar epithelium within 24 h of seed germination, but the aleurone tissue surrounding the starchy endosperm eventually becomes the main tissue expressing this enzyme. The EPB gene family of barley consists of two very similar genes, EPB1 and EPB2, both of which have been …
Generation and characterization of tTS-H4: a novel transcriptional repressor that is compatible with the reverse tetracycline-controlled TET-ON system
2007
Background Conditional gene regulatory systems ensuring tight and adjustable expression of therapeutic genes are central for developing future gene therapy strategies. Among various regulatory systems, tetracycline-controlled gene expression has emerged as a safe and reliable option. Moreover, the tightness of tetracycline-regulated gene switches can be substantially improved by complementing transcriptional activators with antagonizing repressors. Methods To develop novel tetracycline-responsive transcriptional repressors, we fused various transcriptional silencing domains to the TetR (B/E) DNA-binding and dimerization domain of the Tn10-encoded tetracycline resistance operon (TetR (B/E)).…
Regulation ofMUC1Expression in Human Mammary Cell Lines by the c-ErbB2 and Ras Signaling Pathways
2001
The MUC1 protein is a highly O-glycosylated transmembrane molecule that is expressed at the luminal surface of most glandular epithelial cells and is upregulated in carcinomas. Here, we report the effect of the activation of the c-ErbB2 --Ras pathway on the expression of the MUC1 gene in the nontumorigenic mammary cell lines MTSV1-7 and HB2 and in the malignant cell lines T47D and ZR75. Endogenous levels of MUC1 mRNA and protein in HB2 clones permanently overexpressing c-ErbB2 or V12-H-Ras were markedly reduced compared with levels in the parental cell lines. Furthermore, in transient transfection assays, the transcription of a CAT reporter construct driven by the MUC1 promoter was inhibite…
Analysis of expression of the gene encoding for the nuclear autoantigen La/SS-B using reporter gene constructs.
1998
In earlier studies mRNA isoforms encoding for the nuclear autoantigen La were identified. In an alternative La mRNA form the exon 1 was replaced with the exon 1'. Moreover, exon 1' La mRNAs were found to start at different 5'-regions. In dependence on the 5'-start the exon 1' La mRNAs encoded for up to three open reading frames upstream of the La frame, which starts in the exon 2. The exon 1' was located in the intron about 70 nts downstream of the exon 1. The exon 1' La mRNA was proposed to be the result of a promoter switch in combination with an alternative splicing mechanism. The commonly used technique to study the expression of a eucaryotic gene is to fuse a reportergene immediately d…
Sea urchin neural alpha 2 tubulin gene: isolation and promoter analysis
2004
Abstract Expression of Tα2 gene, during sea urchin Paracentrotus lividus development, is spatially and temporally regulated. In order to characterize this gene, we isolated the relevant genomic sequences and scanned the isolated 5 ′ -flanking region in searching for cis -regulatory elements required for proper expression. Gel mobility shift and footprinting assays, as well as reporter gene (CAT and β-gal) expression assays, were used to address cis -regulatory elements involved in regulation. Here we report that an upstream 5 ′ -flanking fragment of PlTα2 gene drives temporal expression of reporter genes congruent with that of endogenous Tα2 gene. The fragment contains cis -elements able to…
Towards light-mediated sensing of bacterial comfort
2013
Abstract Bacterial comfort is central to biotechnological applications. Here, we report the characterization of different sensoring systems, the first step within a broader synthetic biology-inspired light-mediated strategy to determine Escherichia coli perception of environmental factors critical to bacterial performance. We did so by directly ‘asking’ bacterial cultures with light-encoded questions corresponding to the excitation wavelength of fluorescent proteins placed under the control of environment-sensitive promoters. We built four genetic constructions with fluorescent proteins responding to glucose, temperature, oxygen and nitrogen; and a fifth construction allowing UV-induced exp…