Cignal Lenti Reporter Assays
For sensitive assessment of cell signaling activities in virtually any mammalian cell type
Cignal Lenti Reporter Assays are ready-to-transduce lentiviral particles for assessing cell signaling activities in virtually any mammalian cell type.
Cignal Lenti Reporter Assays are highly suited for use in studies of cell signaling in primary cells, stem cells, and difficult-to-transfect cell lines (see figures "Determination of pathway activity in human primary cells", "Notch signaling activity in rat glioma cells", "Measurement of NFkB pathway activity" and "Advantage of using dual luciferase system"). They may also be used in the generation of stable cell lines (see figure "Stable cell line generation") and in high-throughput screening of siRNAs.
Cignal Lenti Reporter Assays 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 or GFP).
Lentiviruses are one of the most effective vehicles to introduce reporter constructs in almost all mammalian cells — including nondividing cells. A transduced lentiviralreporter 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 or GFP reporter activity is modulated and can be measured quantitatively and rapidly.
The Cignal Lenti Reporter Assays 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 include the following:
Cignal Lenti Reporter Assays 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 stable, pathway sensor cell line generation.
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 or GFP) are carried out 18–36 hours post-treatment, depending upon the treatment conditions (see flowchart "Cignal Lenti Reporter Assay procedure").
Stable 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 Lenti Reporter Assays 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.
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