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TCF/LEF Reporter

Luciferase  GFP  Lentiviral luciferase Lentiviral GFP
 
Cignal Lenti TCF/LEF Reporter (GFP) Kit: CLS-018G
The Lenti TCF/LEF Reporter is a preparation of ready-to-transduce lentiviral particles for monitoring the activity of Wnt signaling pathways in virtually any mammalian cell type. The Wnt signaling pathway leads to the dephosphorylation, stabilization, and nuclear translocation of β-catenin. The stabilized β-catenin binds to the TCF/LEF transcription factors, leading to the activation of Wnt-responsive genes. The Lenti TCF/LEF reporter is a preparation of replication incompetent, VSV-g pseudotyped lentivirus particles expressing the GFP gene under the control of a minimal (m)CMV promoter and tandem repeats of the TCF/LEF transcriptional response element (TRE). The reporter has been experimentally optimized to maximize the signal to noise ratio by testing the number of response elements as well as intervening sequence between the response elements.

Using measuring GFP fluorescence, the Lentiviral TCF/LEF Reporter (GFP) can easily and rapidly:

  • Monitor Wnt signaling in cells
  • Study chemical compounds or drugs
  • Analyze phenotypes of RNAi or gene over expression

See the complete lentiviral transcription factor reporter list.

 

Kit Components How It Works Manual & Resources Related products
 
    Lentiviral Control Reporters
Component Specification Concentration* (total volume)
TCF/LEF Reporter - 1 tube An inducible TCF/LEF-responsive firefly luciferase reporter ≥ 0.8 × 107 TU/ml (250 µl)
TCF/LEF Reporter - 8 tube An inducible TCF/LEF-responsive firefly luciferase reporter ≥ 0.8 × 107 TU/ml (2000 µl)
 

Kit Components How It Works Manual & Resources Related Products
 
  • Cignal Technology Overview

  • Brief Protocol

The Cignal Lenti Reporters are ready for transduction right out of the box. There is no need to generate or propagate lentivirus in your laboratory. These vectors are extremely useful for transient transduction studies in difficult to transfect cells or for pathway sensor cell line generation. 

Transient Pathway Regulation Studies in Difficult to Transfect Cells: Target cells are transduced with the Cignal Lenti Pathway Reporter. The cells are typically cultured for 24 to 48 hours to insure lentivirus integration. The cultures are then treated with the biological agents of interest (siRNA, shRNA, chemical compound, viral expression vector, protein, peptide). Reporter assays (firefly luciferase or GFP) are carried out 18 to 36 hours post-treatment, depending upon the treatment conditions.

Pathway Sensor Cell Line Generation: Target cells are transduced with the Cignal Lenti Pathway Reporter. Following transduction, the cells are cultured under puromycin selection to generate a homogenous population of transduced cells. If necessary, single cell cloning may be carried out in order to isolate a clonal pathway sensor cell line. These pathway sensor cell lines serve as a valuable cell-based assay platform, for subsequent screening and mechanism of action studies.

  • Biosafety Features

The Cignal Lenti Reporters are ready-to-transduce, replication incompetent, HIV-based, VSV-G pseudotyped lentiviral particles. The Cignal Lentiviral particles are safe to use. It is recommended that they be treated as Risk Group Level 2 (RGL-2) organisms. Follow all published RGL-2 guidelines for handling and waste decontamination. Details on the requirements for creating a BSL-2 work environment are available in the U.S. Department of Health and Human Services publication Biosafety in Microbiological and Biomedical Laboratories, 4th edition.

The biosafety features engineered into these vectors include:

  • A deletion in the promoter/enhancer region of the U3 portion of 3'LTR ensures self-inactivation of the lentiviral construct after transduction and integration into the genomic DNA of target cells.

  • The Cignal Lentiviral vector and plasmids expressing packaging proteins contain no significant areas of homology, thereby minimizing any chance for recombination.
  • None of the HIV-1 genes (gag, pol, rev) will be expressed in transduced cells, as they are expressed from packaging plasmids lacking packaging signal. Therefore, the lentiviral particles that are generated are replication-incompetent

  • No virulence genes ( vpr, vif, vpu and nef) are present in the Cignal Lentiviral vector.

 
Feature Function
RSV-5' LTR; Hybrid Rous sarcoma Virus (RSV) enhancer/promoter-U5 long terminal repeat Permits viral packaging and reverse transcription of viral mRNA
Psi; Packaging signal Allow viral packaging
RRE; Rev response element Involved in packaging of viral transcript
cppt; Central polypurine tract Involved in nuclear translocation and integration of transduced viral genome
Reporter gene (firefly luciferase or GFP) Allow quantification of transcription
hPGK; human phosphoglycerate kinase eukaryotic promoter Permits high-level expression of the mammalian selection marker (puromycin)
PuroR; puromycin resistance gene Can be used for mammalian selection
SIN/3'LTR; 3' self inactivating long terminal repeat Modified 3'LTR that allows viral packaging but self inactivates the 5'LTR for biosafety purpose. The element also contains a polyadenylation signal for efficient transcription termination
f1 ori; f1 origin of replication Origin of DNA replication for bacteriophage f1
AmpR; ampicillin resistance gene Allows selection of the plasmid in E.coli
TRE; Transcription response element Permits regulation of reporter gene expression by a specific transcription factor
TATA box Act as an minimal promoter

  • Performance Data

Cignal Lenti TCF/LEF reporter (GFP) [5104 TU] transduced around 10,000 293-H cells (24 hours before transduction 5,000 cells were plated per well of 96-well plate). After 24 hours of transduction, medium was changed to complete medium. After 48 hours of transduction, cells were treated with 20 mM (Image B) and 50 mM LiCl (Image C) or were left untreated (Image A). After 24 hours of treatment, bright field and fluorescent images were taken of the cultures transfected with Cignal TCF/LEF-GFP reporter.

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Kit Components How It Works Manual & Resources Related Products
 
Lentiviral Control Reporters Use important positive and negative control lentiviral reporters for best results
User Manuals Cignal Lenti Reporter User Manual (PDF)
White Paper Cignal Reporter Assay Kit: A High Performance Tool for Assessing the Functions of Genes, Biologics and Small Molecule Compounds (PDF)
Web Seminar Attend a live on-line seminar hosted by an Application Scientist about Cignal Reporters
Technical Brochure Cignal Lenti Reporters Technical Brochure (PDF)
Power Point Cignal Lenti Reporters: Measure Signaling Pathway Activity in ANY Mammalian Cell (PDF)
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Kit Components How It Works Manual & Resources Related Products
 
Pathway reporters Complete list of signaling pathway reporters
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10-Pathway Arrays Simultaneously measure the activities of ten (10) signaling pathways in a single experiment
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