QIAGEN Website    Quick Order    Online Seminar    Contact    My Account
Home  >  Products  >  Cignal Reporter Home  >  Lenti Reporter  >  HIF

HIF Reporter

Luciferase  Lentiviral luciferase
 
Cignal Lenti HIF Reporter (luc): CLS-007L
The Lenti HIF Reporter is a preparation of ready-to-transduce lentiviral particles for monitoring the activity of HIF-regulated signaling pathways in virtually any mammalian cell type. The hypoxia-inducible factor-1 (HIF-1) protein is a key regulator of oxygen homeostasis and plays significant roles in cancer progression as well as in cardiovascular diseases. The Lenti HIF reporter is a preparation of replication incompetent, VSV-g pseudotyped lentivirus particles expressing the firefly luciferase gene under the control of a minimal (m)CMV promoter and tandem repeats of the hypoxia response element (HRE). We have experimentally optimized the number of response elements as well as the intervening sequence between response elements to maximize the signal to noise ratio.

Using a luciferase reporter assay, the Lentiviral HIF Reporter (luc) can easily and rapidly:

  • Monitor HIF-regulated signaling pathway activity 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)
HIF Reporter - 1 tube An inducible HIF-responsive firefly luciferase reporter ≥ 0.8 × 107 TU/ml (250 µl)
HIF Reporter - 8 tube An inducible HIF-responsive firefly luciferase reporter ≥ 0.8 × 107 TU/ml (2000 µl)
*exact titer specified on Certificate of Analysis
 

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

Advantage of using Cignal Lenti dual luciferase system: Cignal Lenti HIF-1 reporter (luc) [1.5X105 TU] transduced around 15,000 HepG2 cells (24 hours before transduction 7,500 cells were plated per well of 96-well plate) with and without Cignal Lenti Renilla control (luc) [3X104 TU]. After 42 hours of transduction, medium was changed to assay medium (Opti-MEM + 0.5% FBS + 0.1mM NEAA + 1mM Sodium pyruvate + 100 U/ml penicillin + 100 µg/ml streptomycin). After 48 hours of transduction, cells were treated with 300 µM of CoCl2 for 18 hours. Luciferase or dual-Luciferase assay was performed, and promoter activity values are expressed as arbitrary units using a Renilla reporter for internal normalization. Experiments were done in triplicates, and the standard deviation is indicated. The utilization of Cignal Lenti dual-luciferase system (co-transduction of Cignal Lenti Renilla control along with Cignal Lenti reporter (luc)) improves the fold induction change in hypoxia signaling activity, as normalization of firefly luciferase activity with renilla luciferase activity corrects for cell death caused by the treatment.

Back to Top

 

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 Reporter Technical Brochure (PDF)
Power Point Cignal Lenti Reporters: Measure Signaling Pathway Activity in ANY Mammalian Cell (PDF)
Back to Top
 

Kit Components How It Works Manual & Resources Related Products
 
Pathway reporters Complete list of signaling pathway reporters
Attractene Transfection Achieve high transfection efficiencies and minimal cytotoxicity in your transfections
10-Pathway Arrays Simultaneously measure the activities of ten (10) signaling pathways in a single experiment
shRNA Plasmids Pathway related gene knockdowns - Guaranteed 70% gene knockdown

Back to Top