Cignal™ Lenti Reporters

The Cignal Lenti Reporters are ready-to-transduce lentiviral particles for assessing cell signaling activities in virtually any mammalian cell type. This system utilizes a unique combination of transcription factor reporter technology coupled with lentiviral delivery power. These reporters are powerful tools in functional genomics and drug discovery for assessing pathway activity. When the pathway is activated or inhibited by a drug candidate, gene knockdown (using siRNA), overexpression event (expression vectors), or peptide, luciferase or GFP reporter activity is modulated and can be measured quantitatively and rapidly. The Cignal Lenti Reporters are safe to use in your laboratory.

Why Cignal Lenti Reporters?

• Ready to transduce:
  Lenti reporters arrive as transduction-ready lentiviral particles, not vectors requiring further preparation
  
  
• Transduce virtually any cell type:
  including non-dividing cells, stem cells, and differentiated cells
  
  
• Wide application:
  Use for transient experiments or develop stable cell line reporters

These assays are powerful tools in functional genomics and drug discovery for assessing the biological impact of siRNAs, proteins, and small molecule compounds.

Product Listing and Performance Data

Click here to view the Transcriptional Response Element (TRE) sequences for each reporter assay.

Cignal Lenti Controls:
When conducting experiments using Cignal Lenti Reporters, proper controls are a key element of the experimental design to permit accurate interpretation of reporter assay results and provide assurance of the specificity of the observed response.

How it works

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 Cell Lines: 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.

Performance Data

• Study cell signaling in Primary Cells, Stem Cells, and Difficult to Transfect Cell Lines

Cignal Lenti NFAT reporter determines PKA/Ca 2+ pathway activity in human primary cells (Normal Human Pulmonary Artery Smooth Muscle Cells; PASMC): Cignal Lenti NFAT reporter (luc) [4X10⁵ TU] and Cignal Lenti Renilla control (luc) [1X10⁵ TU] co-transduced around 10,000 PASMC cells (24 hours before transduction 5,000 cells were plated per well of 96-well plate). After 48 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 µ/ml streptomycin). After 54 hours of transduction, cells were treated with 10 ng/ml PMA and 0.5 µM ionomycin for 18 hours. 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.

Cignal Lenti RBP-Jk reporter measures Notch signaling activity in neuronal cells (C6; Rat Glioma Cells): Cignal Lenti RBP-Jk reporter (luc) [2X10⁵ TU] and Cignal Lenti Renilla control (luc) [2X10⁴ TU] transduced around 20,000 C6 cells, rat glioma cells, (24 hours before transduction 10,000 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 infected with 100 MOI of recombinant adenoviruses expressing constitutive active Notch1 (Ad-NICD) or 100 MOI of recombinant adenovirus expressing GFP (Ad-GFP). Dual Luciferase assay was performed 18 hours after infection, 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.

Cignal Lenti NFkB reporter measures NFkB pathway activity in thymocytic cells (D1; Murine T-Cell Leukemia Cells): Cignal Lenti NFΚB reporter (luc) [2.5X10⁵ TU] transduced around 10,000 D1 cells, an IL-7-dependent murine thymocyte cell, (24 hours before transduction 5,000 cells were plated per well of 96-well plate). After 48 hours of transduction, cells were treated with 20 ng/ml of recombinant mouse tumor necrosis factor alpha (hTNFα) protein for 18 hours. Luciferase assay was performed, and promoter activity values are expressed as arbitrary units. Experiments were done in triplicates, and the standard deviation is indicated.

Cignal Lenti SRE reporter measures MAPK/ERK signaling activity in fibroblast cells (CV 1; Normal African green monkey kidney fibroblast): Cignal Lenti SRE reporter (GFP) [5X10⁵ TU] or Cignal Lenti negative control (GFP) transduced around 10,000 CV-1 cells, normal African green monkey kidney fibroblasts, (24 hours before transduction 5,000 cells were plated per well of 96-well plate). 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 10% FBS and 10 ng/ml PMA. After 24 hours of treatment, fluorescent images were taken for non-treated (A) and treated (B) cells transduced with Cignal Lenti SRE reporter (GFP). Then, medium was removed from the wells and fluorescence activity was measured using a fluorometer. The fluorescent activity present in treated and non-treated Cignal Lenti negative control (GFP) transduced cells was subtracted from the fluorescent activity in treated and non-treated Cignal Lenti SRE reporter (GFP) transduced cells and relative fluorescence activities are expressed as arbitrary units. Experiments were done in triplicates, and the standard deviations are indicated.

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.

• Stable Cell Line Generation

Generation of NFkB pathway sensor cell line using Cignal Lenti NFkB reporter (luc): Cignal Lenti NFkB reporter (luc) was used to
develop a stable HEK-293 NFkB sensor cell line for the study of the NFkB signal transduction pathway. The NF-kB sensor cell line was developed by transduction
of HEK-293 cells with a Cignal Lenti NFkB reporter (luc), followed by selection of a clonal population that maintained stable chromosomal integration of the lentiviral vector provirus and responded strongly to stimuli known to activate the NF-kB pathway. The generation of stable HEK-293H NFkB sensor cell line was confirmed by testing the responsiveness of the cell line toward 10 ng/ml of recombinant human tumor necrosis factor alpha (hTNFα) protein after 1st, 5th, 10th and 15th passage of the cell line. Stimulation of the NF-kB pathway by hTNFα results in up to a 100-fold increase in expression of the reporter gene even after two months of culture of NFkB sensor cell line.

• Utilization in HTS (siRNA screening)

Cignal Lenti NFkB Reporter identifies genes regulating NFkB signaling activity using siRNA screen: To determine the suitability of Cignal Lenti reporter for HTS, a pilot scale siRNA screening (42 siRNA screen) was carried out using Cignal Lenti NFkB reporter (luc) to identify genes regulating NFkB signaling activity. The Cignal Lenti reporter showed acceptable Z-factor and coefficient of variation (%CV) in a screen which is comparable to that for the NFkB pathway reporter HEK-293 stable cell line [generated using Cignal Lenti NFkB reporter (luc)], proving the suitability of Cignal Lenti reporters for screening 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.
  
  

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