Cignal Lenti Reporter Assays

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.

Safety

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: 

• A deletion in the promoter/enhancer region of the U3 portion of 3' LTR that 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 express 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
  

 

Features of Cignal Lenti Reporter Assays

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	Allows 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)	Allows 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 reasons, 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	Acts as an minimal promoter

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.