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New Mammalian Two-Hybrid System Detects Protein-Protein Interactions
... protein-protein interactions via the activationof reporter gene expression. This expression occurs as a resul... the assay detects transcriptional activation of a reporter gene (such as luciferase, SEAP, or b -galactosidase) located downstream of the mult...Assess the In Vivo Activation of Signal Transduction Pathways with PathDetect Reporting Systems
...riptional activator GAL4 6,7 ; the pFR-Luc reporter plasmid for expression of the Photinus pyr... When a fusion trans-activator plasmid,the pFR-Luc reporter plasmid and a plasmid encoding an uncharacterized gene arecotransfected into mammalian cells, either......nalyzed for expression of their respective reporter genes. Cells transduced with AdCMV-GFP and Ad-CG (both harboring a GFP reporter gene) were visualized by fluorescence microscopy. Cells transduced with AdCMV-LacZ were stai...Versatile Vectors for Ponasterone A- Inducible Control of Gene Expression in Mammalian Cells
...we performed transient expression assays using the reporter pEGSH-luc in which the coding sequence for... into the MCS of pEGSH. In these experiments, reporter was cotransfected with various amounts of the pERV3 receptor vector, induced with murA, ...Safe Production of High-Titer Retrovirus
...fluorescent protein (GFP)-expressing viral reporter vector pFB-GFP (unpublished); titers were determined by fluorescence- activated cell sorting (FACS). ( Figure 3 ) and are calculated two ways. Titers calculated using percent-shifted values that are linear over two 10-......ormis green fluorescent protein (hrGFP), a reporter protein known for its low cellular toxicity. Expression studies with the hrGFP reporter maintain a healthy cell population and ensure that results are biologically valid. R...Optimizing Transfection Conditions for Studying Signal Transduction Pathways
...s include unique fusion trans-activatorplasmids, a reporter plasmid that encodes the firefly luciferase gene, ...ivate transcription of the luciferase genefrom the reporter plasmid. Luciferase activity that exceeds background levelsindicates that the gene of interest is in...Restriction Endonucleases Undergo Rigorous QC Testing to Ensure Optimal Performance
...ctor ( Figure 3 ), which contains the lacZ reporter gene, is cut at a single position within the multiple cloning site using a five-fold excess of enzyme. A phenol-chloroform extraction is used to purify the linearized vector. The vector is ligated and transformed into Epicuria...Signal Transduction Reporting Systems Using Cis-Acting Enhancer Elements
...f the cis-reporterplasmids contains the luciferase reporter gene driven by a basic promoterelement (TATA box) ... areporter gene in place of the cellular gene. The reporter gene encodes thefirefly luciferase enzyme that can be monitored easily, inexpensively andquantitativ...Stable HeLa Luciferase Reporter Cell Lines Expressing GAL4 Fusion Transactivators
Stable reporter cell lines for signal transduction pathway readout...rans-activatorsbind to the promoter in the pFR-Luc reporter plasmid, which contains five directrepeats of the GAL4-binding element and controls luciferase expre...Versatile Reporter Vectors for Monitoring Viral Transduction
...atagene We describe three new ViraPort retroviral reporter vectors: pFB-hrGFP Y , pFB-Neo-LacZ , ...e excluded selectable markers or readilydetectable reporter genes from the ViraPort retroviral gene expression systemsto ensure that the largest cDNAs possible ...pFB-ERV: Retroviral Delivery of the Ecdysone Receptor Proteins
...re transfected with the ecdysone-inducible reporter vector pEGSH-luc. The following day, cells were in... screen, 24 colonies were transfected with reporter vector, and induced with 10 M ponA or an equivalent volume of vehicle. All 24 of the infecte...Improve Lipid- or Calcium Phosphate-Mediated Transfection of Human Dermal Fibroblasts
...ted with a plasmid expressingthe b -galactosidase reporter molecule under thecontrol of the cytomegalovirus p...d transfection show that expression of the reporter molecule improved over 5-fold after incubating in 10 M Primary ENHANCER reagent and 20-fold ...In Vivo Signal Transduction Pathway Reporting Systems
... of these kinases. When a fusion activatorplasmid, reporter plasmid and uncharacterized gene are cotransfected...ivatetranscription of the luciferase gene from the reporter plasmid. Similarly, theeffects of extracellular stimuli can be studied with these systems. Very lowb...Improved Vectors for PathDetect Trans-Reporting Systems
... yeast GAL4. When a fusion trans-activatorplasmid, reporter plasmid and gene of interest are cotransfected int...cause transcription of theluciferase gene from the reporter plasmid. Luciferase activity, which can beconveniently assayed using Stratagenes Luciferase Assay Ki...A Comparison Study of Lipid Tranfection Reagents in A549, NIH 3T3 and COS-7 Cell Lines
...n reagentto optimize conditions for transfecting a reporter plasmid in three common celllines. In addition, th...nitial optimization step, the signal strength of a reporter gene is measured as the amount of lipid is varied. The reporter plasmid (1.0 g) enco...Versatile Transfection Reagent Offers Low Toxicity and Consistent Performance
...it is standard to measure the signal intensity ofa reporter gene and cellular protein concentration across a m...poTAXI reagent using the b -galactosidase reporter plasmid in HeLa cells showed uncharacteristically low efficiencies. 6 Stratagenes Mycoplasm...Mammalian and Bacterial Expression in One Vector
...n fluorescent protein 5 (GFP) gene as the reporter because its expression can be visualized easily in...re screened by PCR for the presence of the reporter gene. One hundred percent of the screened colonies contained the desired insert, and one of ...Low-Toxicity, Lipid-Mediated Transfection of Mammalian Cells
...omegalovirus (CMV) promoter.Varying amounts of the reporter plasmid were mixed with different volumes ofLipoTA...ion of these cell lines, relative expression ofthe reporter gene was compared 3 to determine the optimalconcentration of DNA and volume of LipoTAXI transfectio...New Reporter Plasmids for Studying Interferon-Stimulated Signal Transduction Pathways
... of theluciferase gene. The pISRE-Luc and pGAS-Luc reporter plasmids monitor theactivation status of signaling...a cis-reporterplasmid that contains the luciferase reporter gene driven by the basic promoterelement (TATA box) joined to direct repeats of cis-acting DNA eleme...Mycoplasma Contamination Reduces the Effect of Lipid-Mediated Transfection of Mammalian Cells
...trations of plasmid DNA that encodes for a reporter gene. Expression of the reporter gene increases until amounts of lipid and/or DNA prove toxic. While performing this ...New Fusion Trans-Activator Plasmids for Studying Signal Transduction Pathways
... unique fusion trans-activator plasmids, a reporter plasmid that encodes the firefly luciferase gene ...on factors. When a fusion trans-activator plasmid, reporter plasmid and uncharacterized gene are cotransfected into mammalian cells, either dire...High-Efficiency Transfections Achieved with New Low-Toxicity Reagent
... characterized. We transfected the pCMVbgal reporter plasmid into HUVEC and HUAEC cells, which are primary cells, with GeneJammer reagent or each of five competitors transfection reagents and compared the results. The best outcomes were obtained in serum-free media. The ...Cis-Reporting System Monitors Intracellular Calcium Mobilization
...ew PathDetect cis-reporter plasmid,the pNFAT-Luc reporter plasmid, is designed for studying the activation o...AT-Luc plasmid ( Figure 1 ) is the pFR-Luc reporter plasmid of the PathDetect trans-reporting system. To this backbone, we replaced the GAL4 bin...Cis-Reporting System Monitors Intracellular Calcium Mobilization
...ew PathDetect cis-reporterplasmid, the pNFAT-Luc reporter plasmid, is designed for studying the activationof...AT-Luc plasmid ( Figure 1 ) is the pFR-Luc reporter plasmid of the PathDetect cis-reporting system. To this backbone, we replaced the GAL4 bindi...Genetic reporter systems have developed into an essential tool for ...moter, enhancer) is cloned together with the reporter gene in an expression vector, which is subsequently used to transfect cells. Quantification of...Genetic reporter systems have developed into an essential tool for ...moter, enhancer) is cloned together with the reporter gene in an expression vector, which is subsequently used to transfect cells. Quantification of...TransMessenger Transfection Reagent Handbook
... with a chloramphenicol acetyltransferase (CAT) reporter RNA are typically incubated for approx... Include positive controls to ensure that the reporter assay is working properly. Serum Serum ma...Transfection Reagent Selector Kit Handbook
...mple, cells transfected with -gal or cat reporter constructs are typically incubated for 2448 h afte...fection to obtain maximal expression levels of the reporter gene. Transfection Optimization The selector pro...Uses and Applications of X-tremeGENE Q2 Transfection Reagent
...e those recommended by the suppliers. The level of reporter gene expression was determined 48 hours post transfection, using the beta-Gal Reporter Gene Assay Kit (Roche Applied Science, Cat. No. 1 758 241). Note: The level of expression of a transfected gene will vary with the specific cel...General Considerations for Successful Transfection Experiments
...abolic end products (e.g., pH). The extent of reporter gene expression is directly correlated with the cell number at the beginning of transfection and the growth rate prior to cell lysis....Uses and Applications of FuGENE 6 Transfection Reagent
...ider repeating the experiment with a secreted reporter gene assay such as SEAP, hGH, or a standard -gal control vector. Cells secreting SEAP should show little to no evidence of cytotoxicity. The culture may be contaminated with mycoplasma Use the Myco...Uses and Applications of FuGENE 6 Transfection Reagent
...ake sure you are able to measure the expression of reporter gene or transfected protein. Plasmids with reporter genes are available from Roche Applied Science. Handle the DNA as aseptically as possible to avoid ...Tools for Proteomic Science II
...a broad line of sensitivekits and reagent sets for reporter gene assays on transfectedcells. These nonradioactive products measure promotoractivity with a sensitivity equal to or greater thanthat of radioisotopic assays, providing a safe alternativewithout sacrificing sensitive results. Apo......an cells using green fluorescent protein(GFP) as a reporter molecule. Transfection of CHO-K1 cells withexpress...eived andare expressing the foreign DNAsequence, a reporter gene can beused. The reporter gene can bepresent on the same vector as thegene of interest or can be......or measuringthe transfection efficiencyusing a GFP reporter plasmid inprimary cells on the 2100 bioanalyzer.Great emphasis was placedon being able to perform theanalysis with a minimum of cellsand to eliminate steps in the protocolwhere cells may be lost.These protocols facilitate working with very few cells....Cancer-Related miRNAs Uncovered by the mirVana miRNA Microarray Platform
... approaches are far more difficult, reporter constructs have been used to sho...els that are disproportionate to reporter vector controls that contain mutations in the putative miRNA binding sites [10]. I...Combat RNase Contamination in the Lab
...strate tagged with a fluorescent reporter molecule (fluor) on one end and a quencher on the other. In the absence of RNases, the physical proximity of the quencher dampens fluorescence from the fluor to extremely low levels. When RNases a...Isolate It All: siRNA miRNA Total RNA Native Protein
... protein (e.g. functional assay, reporter activity). For RNA isolation, the HeLa cell lysate was immediately mixed with an equal volume of 2X Denaturing Solution. This solution contains a high concentration of chaotropic denaturant, resul...pSilencer 4.1-CMV: Versatile Vectors for Expression of siRNA, miRNA, and mRNA
...ells. As shown in Figure 3, high levels of reporter protein were readily detected in the transfected cells 24 or 48 hours after transfection. The presence of a hygromycin, neomycin, or puromycin antibiotic resistance genes on each p Silencer 4.1-CMV vector further allows for s...MicroRNA Profiling by Array Analysis Reveals Critical BioMarkers
...get protein expression and using reporter constructs [15, 16]. Three critical points about miRNA function are: (1) Each miRNA appears to regulate the translation of multiple genes, and many genes appe...Getting Started with MicroRNA Research
...nts from phenotypic responses or reporter assays. MicroRNA activity can be ...is a highly sensitive luciferase reporter vector that is useful for studying interactions between miRNA and target sites fro...Precursor miRNAs for Successful miRNA Functional Studies
...To test for strand activation, a series of reporter plasmids were produced using the pMIR-REPORT miRNA...in cells transfected with a combination of reporter vector and six different variants containing the let-7b or miR-33 miRNA sequences are shown ...Reagents for Up and Down-Regulation of miRNA Activity in Mammalian Cells
... Using an miRNA target containing a luciferase reporter to monitor miRNA activity, we tested three...cursor miRNA designs resulted in decreased reporter activity, with Design #3 providing the strongest miRNA activity (i.e. inhibition of a target...Recommendations for Successful siRNA Library Screens
...ays (e.g. ELISA, enzymatic activity assay, reporter signal measurements) must be normalized by the number of cells in each well. Panel B . HeLa cells were transfected as described in Panel A. Caspase 3 activity was monitored as described in Figure 3. Green arrows note genes th...siRNA Design: It's All in the Algorithm
...ch as silencing of cotransfected exogenous reporter constructs. In collaboration with Ambion, Cenix BioScience has chosen the more rigorous path of directly measuring the effectiveness of approximately 1,100 of its own algorithm-designed siRNAs in silencing almost 400 endogenou...The let-7 microRNA Family Regulates RAS: Implications for Development and Oncogenesis
...1]. Using genetic approaches and reporter constructs with the let-60/RAS ...ter. When transfected into HeLa cells, the reporter constructs with the RAS 3'UTRs expressed 4- to 8-fold less luciferase activity than the cont...RNA Interference and Gene Silencing: History and Overview
...epicker A. (1994) Posttranscriptional silencing of reporter transgenes in tobacco correlates with DNA methylation. Proc Natl Acad Sci USA 91 : 10502-10506. (<A href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=7937983&dopt=Abstract">Abstrac...Commercial Plasmid Preparation Methods Yield DNA With Different Transfection Capabilities
... and used in a microplate chemiluminescent reporter assay for the detection of b-galactosidase activity according to the manufacturers specifications (Galacto-Light Plus, Tropix, Inc.). Enzyme activity was measured using a LumiCount microplate luminometer (Packard Instrument Co...Real-Time Multiplex PCR from Genomic DNA Using the iCycler iQ Detection System
... real-time quantitative PCR, a fluorescent reporter molecule is added to the chemical reaction. This reporter may be specific for the desired amplification product, or it can be nonspecific, use......mance. At varying degrees of misalignment, reporter channel performance was evaluated using th...e parameters listed in Table 1. The reporter validation test is designed to confirm that the reporter channel is correctly measuring assa...Selection of Standards for Bio-Plex Cytokine Assays, Rev A
... PMT. The PMT detects assay signals in the reporter or RP1 channel. The PMT voltage on the Bio...ne concentrations are expected. The voltage of the reporter PMT may also be varied to maximize assay performance. The high-sensitivity standards......to be visualized by monitoring fluorescent reporter molecules in the PCR. In this way the exponential phase of the amplification can be determined. The point at which fluorescence significantly increases above background is described as the threshold cycle (C T )). At the C T )...The Gradient Feature: Use in Optimization of Allelic Discrimination Assays, Rev B
...are labeled at the 5' end with a different reporter dye such as FAM, HEX, TET, Cy5, Texas Red, etc. for each ASO probe. For the analysis of a biallelic genetic locus, two allele-specific probes are synthesized: a probe specific for the detection of allele 1 labeled with one dye...The iCycler iQ Detection System for TaqMan Assays, Rev B
... probe is labeled at the 5' end with a fluorescent reporter molecule such as fluorescein and at the 3'...rivative, which acts as a quencher for the reporter (Heid et al. 1996). When the two fluorophores are fixed at opposite ends of the 2030 nt prob......R products. In real-time PCR, a fluorescent reporter molecule is used to track the progress of ... Many systems exist to introduce the fluorescent reporter molecule to the reaction. The Amplifluor universal amplification and detection system ......35S promoter (Kay et al., 1987) to the GUS reporter gene creates a gene construct (D35S) that expresses GUS constitutively in transformed plant tissues. Inclusion of this construct is used as an internal control to reduce variability in all our particle bombardment experiments ...Biolistic Gene Transfer to Generate Transgenic Schistosomes, Rev A
...reen Fluorescent Protein (GFP) gene as the reporter gene. Parasite Stock and Culture C... culture conditions until heat shock to induce reporter gene activity. After this treatment, the survival rate decreased below 50%. Light microscopy...... Results and Discussion Detection of reporter gene activity in vitro. HeLa cell monolayers ... cells was almost not detectable. By analyzing the reporter gene activity of both plasmids used, we were able to show that the Helios gene gun-mediated ...Single-Cell Complementation of Barley Mlo Mutants Using a PDS-1000/He Hepta System
...n delivery of a GUS (β-glucuronidase) reporter gene construct. Application of the Hepta a...was used. In this plasmid the β-glucuronidase reporter gene is driven by a double constitutive cauliflower mosaic virus 35S promoter. Prepa...Other ContentsmetabolicmetabolicmetabolicmetabolicmetabolicmetabolicmetabolicmetabolicmetabolicmercurymercurymercurymercurymetabolizemetabolizemetabolizemetabolizemetabolizemetabolizemetacarpalmetastaticmetastaticmetastaticmetastaticmetastaticmetastasismetastasismetastasismetastasisOther TagsshedshedshedshedChannelChannelChannelChannelIonIonIonIonIonIonIonIonIonIonRegulateRegulateyearyearyearyearsoilLowLowLowLowLowLowLowLowdroughtsuggestssuggestssuggestsNeedNeedNeedNeedOther Tagspremium 2premium 3sub 2anything 2anything 3anything 4anything 5anything 6anything 7anything 8anything 9anything 10beings 2beings 3beings 4beings 5neutralizing 2interactive 2
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