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Results for “Reporter assay”

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At least 55 records · Page 3Linked to original sources
PubMed1987

An international collaborative study on the performance of protein C antigen assays. Report of the ICTH subcommittee on protein C. International Committee on Thrombosis and Haemostasis.

An international collaborative study was undertaken to evaluate the performance and specificity of protein C antigen (PC) assays. Thirteen lyophilized plasma samples were distributed among 17 laboratories and analysed with 24 methods. No statistically significant results were obtained with the different methods in plasmas containing only the protein C zymogen. ELISA's, RIA's and IRMA's were found to be more sensitive than the electro-immunoassay. In plasmas of patients on oral anticoagulant treatment ELISA methods tend to give lower PC antigen levels than the electro-immunoassay. Complexes between activated protein C (APC) and the protein C inhibitor (PCI), when present in plasma together with PC zymogen, are detected with 100% efficiency in the electro-immunoassay, with 50% efficiency in the ELISA, and with less than 10% efficiency in the RIA or in assays using monoclonal antibodies against PC. Mean coefficient of variation was calculated to be 22%, and could be reduced - especially in case of the ELISA by normalisation. Within laboratory variation was calculated to be 11.7% and between laboratory variation 17.8%.

Administration, Oral↗
PubMed2005

Evaluation of an FRDA-EGFP genomic reporter assay in transgenic mice.

Friedreich ataxia is an autosomal recessive neurodegenerative disorder caused by a GAA trinucleotide expansion in the first intron of the Friedreich ataxia gene (FRDA) that causes reduced synthesis of frataxin, a mitochondrial protein likely to be involved in biosynthesis of iron-sulfur clusters. This leads to increased oxidative stress, progressive loss of large sensory neurons, and hypertrophic cardiomyopathy. To elucidate the mechanisms regulating FRDA expression and to develop an in vivo assay for agents that might upregulate FRDA expression in a therapeutically relevant manner, we have generated transgenic mice with a BAC genomic reporter construct consisting of an in-frame fusion between FRDA and the gene coding for enhanced green fluorescent protein (EGFP). Production of full-length frataxin-EGFP fusion protein was demonstrated by immunoblotting. EGFP expression was observed as early as day E3.5 of development. Most tissues of adult transgenic mice were fluorescent. The level of FRDA-EGFP expression in peripheral blood, bone marrow, and cells obtained from enzymatically disaggregated tissues was quantitated by flow cytometry. There was a twofold increase in EGFP expression in mice homozygous for the transgene when compared to hemizygous mice. These transgenic mice are a valuable tool for the examination of spatial and temporal aspects of FRDA gene expression and for the preclinical evaluation of pharmacological inducers of FRDA expression in a whole-animal model. In addition, tissues from these mice should also be valuable for stem cell transplantation studies.

Animals↗
PubMed2006

Reporter assay systems for [URE3] detection and analysis.

The Saccharomyces cerevisiae prion [URE3] is the infectious amyloid form of the Ure2p protein. [URE3] provides a useful model system for studying amyloid formation and stability in vivo. When grown in the presence of a good nitrogen source, [URE3] cells are able to take up ureidosuccinate, an intermediate in uracil biosynthesis, while cells lacking the [URE3] prion can not. This ability to take up ureidosuccinate has been commonly used to assay for the presence of [URE3]. However, this assay has a number of practical limitations, affecting the range of experiments that can be performed with [URE3]. Here, we describe recently developed alternative selection methods for the presence or absence of [URE3]. They make use of the Ure2p-regulated DAL5 promoter in conjunction with ADE2, URA3, kanMX, and CAN1 reporter genes, and allow for higher stringency in selection both for and against [URE3], nonselective assay of prion variants, and direct transformation of prion filaments. We discuss advantages and limitations of each of these assays.

Amino Acid Transport Systems, Basic↗
PubMed2000

A fluorescent reporter assay for the detection of ligands acting through Gi protein-coupled receptors.

Accompanying the advances in basic biology of G protein-coupled receptors (GPCRs) is the practical need among biopharmaceutical companies for sensitive assays to assess GPCR function, particularly formats that are compatible with high-throughput drug screening. Here we describe a novel cell-based assay format for the high-throughput detection of ligands for Gi protein-coupled receptors. Two Gi-GPCRs, mu-opioid receptor (mu-OPR) and 5-hydroxytryptamine receptor la (5HT1aR) are employed as model receptor targets. The key feature of this assay system is the isolation of stable, clonal Chinese hamster ovary (CHO) cell lines that carry three separate expression plasmids: (1) a chimeric Gq/i5 protein (which re-directs a negative Gi-type signal to a positive Gq-type response), (2) a given Gi-GPCR, and (3) a beta-lactamase (beta1a) reporter gene responsive to Gi-GPCR signaling. Cell-based assays built using this format show appropriate rank order of potency among a reference set of receptor agonist and antagonist compounds. Such assays are also robust, reliable, and can be used for industrial-scale applications such as high-throughput screening for drug leads.

Animals↗
PubMed2000

Transcriptional activation analysis using bioluminescent reporter assays.

Analysis of promoter and enhancer DNA sequences provides the researcher with valuable information regarding the expression patterns of genes. Insertion of small DNA fragments containing the regulatory sequence of interest into vectors carrying reporter genes allows for the accurate quantitative analysis of the gene's expression patterns and responses to various stimuli. The use of bioluminescent reporter genes provides a simple, rapid, and inexpensive system that generates virtually no toxic or radioactive waste products. In addition, bioluminescent reporter vectors are more sensitive than previous methods such as the chloramphenicol acetyl transferase (CAT) systems, that require the use of hazardous chemicals and isotopically labeled reagents.

Animals↗
PubMed2005

Development of a versatile reporter assay for studies of retinol uptake and metabolism in vivo.

The two isomers of retinoic acid (RA), all-trans RA and 9-cis RA, are produced in several tissues in order to allow specific control of target gene transcription. Given the high potency of these receptor ligands, it seems likely that the cellular uptake and metabolic activation of the precursor, retinol (vitamin A), should be a highly regulated process. Several retinol dehydrogenases and components involved in the downstream events have been identified and partially characterized. However, less is known about the cellular uptake of retinol, and the isomerase activity giving rise to the 9-cis and 11-cis branches of the pathway. In this work, we show that the 9-cis RA biosynthesis pathway can be fully reconstituted in cultured HEK293A cells expressing a reporter system, including an endogenous isomerase activity converting all-trans retinol into 9-cis retinol. This assay allows for functional studies of known components, as well as screening for yet unidentified genes involved in the pathway. In addition to free all-trans retinol, we find that these cells can take up retinol from plasma retinol binding protein (RBP) by a mechanism that can be efficiently inhibited by blocking antibodies, suggesting that the uptake may involve a cellular receptor. We also demonstrate that overexpression of CRBPI can drive the accumulation of intracellular retinol from unbound retinol added to the medium. Thus, this versatile cellular assay can be used to study several aspects of retinol uptake and metabolism in vivo.

Alitretinoin↗
PubMed1999

Measurement of responses from Gi-, Gs-, or Gq-coupled receptors by a multiple response element/cAMP response element-directed reporter assay.

We have established a rapid, sensitive, high-throughput assay that requires one assay condition to detect agonist effects from Gi-, Gs-, and Gq-coupled receptors. We utilized a vector containing a promoter with three multiple response elements, the vasoactive intestinal peptide promoter and a cAMP response element controlling the transcription of the luciferase gene. An adrenergic agonist, para-aminoclonidine, inhibited forskolin-stimulated luciferase expression when cells were cotransfected with the Gi-coupled alpha(2)-C adrenergic receptor and the MRE/CRE reporter vector. Further, we demonstrate that gastrin-releasing peptide, which activates a Gq-coupled GRP receptor, isoproterenol, which activates a Gs-coupled beta-adrenergic receptor, calcium ionophores, and phorbol 12-myristate 13-acetate, a stimulator of protein kinase C, can mediate increases in luciferase expression in the presence of forskolin but not in its absence. The effect at Gi-coupled receptor activation correlates with the phosphorylation of the CRE binding protein (CREB); however, the mechanisms mediating the responses to Gq- and Gs-coupled receptors are more complex. We demonstrate that this assay is useful for pharmacological analysis of both agonists and antagonists and has the potential to associate orphan G-protein-coupled receptors with their corresponding ligands.

Base Sequence↗
PubMed1994

A cell-based reporter assay for the identification of protein kinase C activators and inhibitors.

Transmission of extra cellular signals across biological membranes results in the generation of lipid metabolites which in turn influence specific cellular events such as cell growth or differentiation. Many of these lipid messengers can activate protein kinase C (PKC) isozymes of which one function is to perpetuate the extracellular signals to the nucleus by phosphorylating other targets proteins. We have engineered mammalian cell lines to identify and evaluate activators and inhibitors of PKC-dependent and independent signal transduction pathways. The A31 mouse fibroblast cell line, has been stably transfected with a construct containing a triplet repeat of the TPA response element (TRE) upstream of a thymidine kinase promoter fused to the human growth hormone (hGH) gene. A31 cells containing this reporter construct exhibit significant increases in hGH secretion following stimulation by phorbol esters or other mitogens. The levels of hGH secretion are modulated in this system using different pharmacological agents. We demonstrate that this assay can be used to identify specific and general inhibitors as well as activators of the signal transduction pathway mediated by PKC isozymes.

Animals↗
PubMed2006

Cardiovascular expression of the mouse WNK1 gene during development and adulthood revealed by a BAC reporter assay.

Large deletions in WNK1 are associated with inherited arterial hypertension. WNK1 encodes two types of protein: a kidney-specific isoform (KS-WNK1) lacking kinase activity and a ubiquitously expressed full-length isoform (L-WNK1) with serine threonine kinase activity. Disease is thought to result from hypermorphic mutations increasing the production of one or both isoforms. However, the pattern of L-WNK1 expression remains poorly characterized. We generated transgenic mice bearing a murine WNK1 BAC containing the nlacZ reporter gene for monitoring L-WNK1 expression during development and adulthood. We observed previously unsuspected early expression in the vessels and primitive heart during embryogenesis, consistent with the early death of WNK1(-/-) mice. The generalized cardiovascular expression observed in adulthood may also suggest a possible kidney-independent role in blood pressure regulation. The second unsuspected site of L-WNK1 expression was the granular layer and Purkinje cells of the cerebellum, suggesting a role in local ion balance or cell trafficking. In the kidney, discordance between endogenous L-WNK1 and transgene expression suggests that either cis-regulatory elements important for physiological renal expression lie outside the BAC sequence or that illegitimate interactions occur between promoters. Despite this limitation, this transgenic model is a potentially valuable tool for the analysis of spatial and temporal aspects of WNK1 expression and regulation.

Alternative Splicing↗
PubMed1998

Translation efficiencies of the 5' untranslated region from representatives of the six major genotypes of hepatitis C virus using a novel bicistronic reporter assay system.

The 5' untranslated region (5'UTR) of hepatitis C virus (HCV) contains an internal ribosome entry site (IRES) which directs translation of the viral open reading frame (ORF). The 5'UTR is highly conserved between virus isolates in both primary sequence and predicted secondary structure. We cloned and sequenced the 5' regions (nt 18 of the 5'UTR to nt 15 of the core coding sequence) of HCV isolates representing the six major genotypes and subcloned these into a bicistronic, dual luciferase reporter construct. The relative expression of the two luciferases, one directed by the HCV IRES and the other by cap-dependent ribosome scanning, was used to compare the activities of the different IRES elements in transfected cells. The 5'UTR from a genotype 2b isolate was the most efficient at directing translation in all four cell lines tested: BHK-21, HeLa-T4+, HuH7 and HepG2. In HepG2 cells the 2b 5'UTR was three times as efficient as the type 6a 5'UTR, which was generally the least active IRES tested. These data suggest that HCV isolates are not able to translate their ORF with equal efficiency, and provide a starting point from which further sequence-function studies can be undertaken.

5' Untranslated Regions↗
PubMed1995

Clinical validation of the new ELSA-CA 125 II assay: report of a European multicentre evaluation.

The ELSA-CA 125 II is a second-generation radioimmunoassay for the quantification of CA 125 in serum. In a multicentre study involving 49 follow-ups of patients with ovarian cancers, and 880 other patients, 2.8% of healthy persons, 25% of 149 patients with benign gynaecological diseases and 39% of 82 patients with benign non-gynaecological diseases had CA 125 levels above 35 U/ml. Using the 35 U/ml cut-off, sensitivities among epithelial ovarian cancers were found to be 85% in serous tumors, 41% in mucinous tumors and 83% in other types. During follow-up of patients with serous ovarian cancers, we observed an equivalent behaviour of both assays--first- and second-generation--with the clinical evolution. We also compared results obtained with other assays commercially available; these were significantly different when a polyclonal antibody was used in the sandwich assay.

Adolescent↗
PubMed1996

Accelerating the pace of luciferase reporter gene assays.

Luciferase reporter gene assays have gained more importance because of their easy readout, high sensitivity and lack of environmental waste disposal problems. However, several obstacles remain that have prohibited a wider use and the implementation of this type of assay in high-throughput screening programs: (i) Measurements need to be carried out within an active enzyme reaction, and the assessment of such reactions are time-dependent; (ii) the signal produced has a "flash" type characteristic and therefore requires specialized equipment for measurement; and (iii) side-reactions can occur that interact with the signal readout of the assay in a non-reproducible way. These hurdles make an otherwise convenient assay principle troublesome for larger-scale screening use. We have attempted to overcome these problems by different means, leading to the development of LucLite, a stable signal homogeneous reagent system. This system allows use in a higher throughput screening capacity and enables the use of standard scintillation/luminescence instruments.

Biological Assay↗
PubMed2000

Development of a facile method for high throughput screening with reporter gene assays.

This report describes a facile methodology for high throughput screening with stable mammalian cell reporter gene assays. We have adapted a 96-well adherent cell method to an assay in which cells propagated in suspension are dispensed into 96- or 384-well plates containing test compounds in 100% DMSO. The validation of a stable CHO cell line that expresses 6xCRE-luciferase for use as a reporter gene host cell line is described. The reporter gene, when expressed in this particular CHO cell line, appears to respond specifically to modulation of cAMP levels, thus the cell line is appropriate for screening and pharmacological analysis of Galpha(s)- and Galpha(i)-coupled seven-transmembrane receptors. The development of the new suspension cell assay in both 96- and 384-well formats was performed using a derivative of the CHO host reporter cell line that was stably transfected with human melanocortin-1 receptor. The response of this cell line to NDP-alpha-melanocyte-stimulating hormone and forskolin was nearly identical between the adherent and suspension methods. The new method offers improvements in cost, throughput, cell culture effort, compound stability, accuracy of compound delivery, and hands-on time. The 384-well assay can be performed at high capacity in any laboratory without the use of expensive automation systems such that a single person can screen 100 plates per day with 3.5-4 h hands-on time. Although the system has been validated using Galpha(s)-coupled receptor-mediated activation of a cAMP response element, the method can be applied to other types of targets and/or transcriptional response elements.

Animals↗