Cignal Finder Lenti Reporter Arrays

Each array includes 10 Cignal Lenti Reporter Assays and 2 controls in transduction ready tube format. Cignal Finder Lenti Reporter Arrays utilize a unique combination of transcription factor reporter technology coupled with lentiviral delivery.

Cignal Lenti Reporter Assays consist of multiple repeats of a specific transcription factor’s binding site and basic promoter elements to drive the expression of a reporter gene (firefly luciferase).

Lentiviruses are one of the most effective vehicles to introduce reporter constructs in almost any mammalian cells — including nondividing cells. A transduced lentiviral reporter construct is integrated into cellular genomic DNA and provides stable, long-term expression of a reporter gene.

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 reporter activity is modulated and can be measured quantitatively and rapidly.

Safety

Cignal Lenti Reporter Assays contained in the Cignal Finder Lenti Reporter Arrays are ready-to-transduce, replication incompetent, HIV-based, VSV-G pseudotyped lentiviral particles (see figure "Cignal Lenti Reporter Assay" and table "Features of Cignal Lenti Reporter Assays"). 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 includes the following:

• 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 Lenti Reporter Assay and plasmids expressing packaging proteins contain no significant areas of homology, minimizing any chance for recombination 
• None of the HIV-1 genes (gag, pol, rev) are expressed in transduced cells, since they are expressed from packaging plasmids lacking packaging signal. Therefore, the lentiviral particles generated are replication-incompetent 
• No virulence genes (vpr, vif, vpu, and nef) are present in the Cignal Lenti Reporter Assay

Cignal Finder Lenti Reporter Arrays are immediately ready for transduction, without the need to further generate or propagate lentivirus. These vectors are highly suited for transient transduction studies in difficult to transfect cells or for pathway sensor cell line generation (see flowchart "Cignal Lenti Reporter procedure").

The procedure is comprised of 3 simple steps:

• Transduce Cignal Finder Reporter Arrays and test nucleic acids into cells
• Treat with siRNA, protein, peptide, or small molecule of interest 
• Perform reporter quantitation using luciferase activity assays

Transient pathway regulation studies in difficult-to-transfect cell lines

Target cells are transduced with the Cignal Lenti Reporter Assay. The cells are typically cultured for 24–48 hours to ensure lentivirus integration. The cultures are then treated with the biological agents of interest (siRNA, shRNA, chemical compound, viral expression vector, protein, or peptide). Reporter assays (firefly luciferase) are carried out 18–36 hours after treatment, depending upon the treatment conditions.

Pathway sensor cell line generation

Target cells are transduced with the Cignal Lenti Reporter Assay. Following transduction, the cells are cultured with 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.

Cignal Finder Lenti Reporter Arrays are powerful tools in functional genomics and drug discovery for assessing cell signaling activities in virtually any mammalian cell type. They are highly suited for assessing the biological impact of siRNAs, proteins, and small molecule compounds.