Would you like to pinpoint the signaling pathways regulated by your gene product or compound, in cell types not amenable to high efficiency transfection? If so, consider Cignal Finder Lenti Reporter Arrays, the first commercial products that enable a comprehensive analysis of multiple pathways (up-to ten) in difficult-to-transfect cells. This system utilizes a unique combination of transcription factor reporter technology coupled with lentiviral delivery power.

Why Cignal Finder Lenti 10-Pathway Reporter Arrays?

• Comprehesive biology: Profile changes in the activities of ten signaling pathways relevant to a specific biological process
• Transduce any cell type:
  Transduce virtually any cell type, including non-dividing cells, stem cells, and differentiated cells
• Ready to transduce:
  Lentiviral vectors arrive as transduction-ready lentiviral particles, eliminating any need to construct and amplify lentivirus in your laboratory

Which ten pathways? See the Cignal Finder Array pathway lists

The Cignal Finder 10-Pathway Reporter Arrays are ideally suited for discovering:

• siRNA/shRNA/miRNA phenotypes
• Biological responses to chemical compounds
• Mechanisms of action of proteins, peptides, and ligands

Available Cignal Finder 10-Pathway Reporter Arrays (luciferase)

How It Works

Each Lenti Reporter Array includes 10 Cignal Lenti Pathway Reporters and two controls. All reporters and controls are delivered as transduction-ready lentiviral particles (250µl at 1X107 transducing units/ml). All reporter assays utilize firefly luciferase reporter gene technology.

Firefly luciferase results are calculated for each transduced culture. The change in the activity of each signaling pathway is determined by comparing the normalized luciferase activities of the reporter in treated versus untreated cells.

The identically treated negative control expresses firefly luciferase under the control of a minimal promoter and serves as a pathway specificity control. The positive control is a valuable reagent for optimizing your transduction conditions, by monitoring GFP expression.

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.

Simple Procedure:

• Transduce Cignal Lenti Reporters into target cells
• Treat with nucleic acid, protein, peptide, or small molecule of interest
• Perform reporter quantitation using firefly luciferase activity assay

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	Allows episomal replication of plasmid in eukaryotic cells
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

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