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Cignal Lenti Reporter Assays

For sensitive assessment of cell signaling activities in virtually any mammalian cell type

  • Transduce virtually any mammalian cell type
  • Transduce nondividing cells, stem cells, and differentiated cells
  • Use for transient experiments or develop stable cell line reporters
  • Transduction-ready lentivirus requires no further preparation

Cignal Lenti Reporter Assays are ready-to-transduce lentiviral particles for assessing cell signaling activities in virtually any mammalian cell type.

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Cignal Lenti Reporter Assays are intended for molecular biology applications. These products are not intended for the diagnosis, prevention, or treatment of a disease.


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Determination of pathway activity in human primary cells.
The Cignal Lenti NFAT Reporter Assay was used to determine PKA/Ca2+ pathway activity. Cignal Lenti NFAT Reporter Assay (luc) (4 x 105 TU) and Cignal Lenti Renilla Control (luc) (1 x 105 TU) cotransduced around 10,000 normal human pulmonary artery smooth muscle cells (PASMC). After 48 hours transduction, medium was changed to assay medium. After 54 hours 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 performed in triplicate and the standard deviation is shown.
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Notch signaling activity in rat glioma cells.
The Cignal Lenti RBP-Jk Reporter Assay was used to measure Notch signaling activity in C6 (rat glioma) cells. Cignal Lenti RBP-Jk Reporter Assay (luc) (2 x 105 TU) and Cignal Lenti Renilla Control (luc; 2 x 104 TU) transduced around 20,000 C6 cells. After 24 hours transduction, medium was changed to complete medium. After 48 hours 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 performed in triplicate and the standard deviation is shown.
2

Measurement of NFκB pathway activity.

The Cignal Lenti NFκB Reporter Assay was used to measure NFκB pathway activity in thymocytic cells (D1; Murine T-cell leukemia cells). Cignal Lenti NFκB Reporter Assay (luc) (2.5 x 105 TU) transduced around 10,000 D1 cells. After 48 hours transduction, cells were treated with 20 ng/ml recombinant mouse TNFα (hTNFα) protein for 18 hours. Luciferase assay was performed and promoter activity values are expressed as arbitrary units. Experiments were performed in triplicate and the standard deviation is shown.
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Advantage of using dual luciferase system.
Cignal Lenti HIF-1 Reporter (luc) (1.5 x 105 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 (3 x 104 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 the dual-luciferase system (cotransduction of Cignal Lenti Renilla Control along with Cignal Lenti Reporter Assay (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.
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Stable cell line generation
The Cignal Lenti NFkB Reporter Assay (luc) was used to generate an NFkB pathway sensor cell line for the study of the NFkB signal transduction pathway. The NFkB sensor cell line was developed by transduction of HEK-293 cells with the Cignal Lenti NFkB Reporter Assay (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 NFkB pathway. The generation of stable HEK-293 NFkB sensor cell line was confirmed by testing the cell line with 10 ng/ml of recombinant hTNFα after 1, 5, 10, and 15 passages of the cell line. Stimulation of the NFkB pathway by hTNFα results in up to a 100-fold increase in expression of the reporter gene even after 2 months of cell culture.
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Cignal Lenti Reporter Assay procedure.
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Cignal Lenti Reporter Assay.
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Cignal Lenti Reporter Assays.
Performance
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.
Principle

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
FeatureFunction
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

Procedure

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.

Applications

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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Brochures & Guides (1)
For measurement of signaling pathways in any mammalian cell
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Kit Handbooks (1)
For lentiviral-based cell signaling activity assays
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VDRE Reporter Assay (luc) Cignal Lenti Reporter Assays
Introduction
The Cignal Lenti Reporter Assays are self-inactivating replication incompetent viral particles. The lentiviral particles are ready to add directly to your cells. The Lenti VDRE Reporter is a preparation of ready-to-transduce lentiviral particles for monitoring the transcriptional activity of the Vitamin D receptor (VDR) in virtually any mammalian cell. VDR is a member of the steroid receptor superfamily. In its ligand bound state, VDR forms heterodimers with RXR and regulates gene expression by binding to specific hormone response elements. The Lenti VDRE reporter is a preparation of replication incompetent, VSV-g pseudotyped lentivirus particles expressing the firefly luciferase gene under the control of a minimal (m)CMV promoter and tandem repeats of the VDR transcriptional response element (TRE). We have experimentally optimized the number of response elements as well as the intervening sequence between response elements to maximize the signal to noise ratio. Using a simple luciferase assay, you can easily monitor the activity of VDR-mediated signaling pathways and determine the effect of various treatments, such as gene knockdown, over-expression, and chemical compounds on those pathways.


The Cignal Lenti VDRE Reporter Assay measures VDRE transcription activity. The Cignal Lenti VDRE Reporter (luc) [5 x 105 TU] and Cignal Lenti Renilla control (luc) [2.5 x 104 TU] co-transduced approximately 104 HeLa cells (24 hours before transduction, 5000 cells were plated per well of a 96-well plate). After 18 hours of transduction, medium was changed to complete growth medium. After 42 hours of transduction, medium was changed to assay medium (Opti-MEM containing 0.5% FBS and 1% NEAA). After 48 hours of transduction, cells were treated with 0.05 µM calcitriol for 18 hours. A luciferase assay was performed 66 hours after transduction, and promoter activity values are expressed as arbitrary units. Experiments were performed in triplicate, and the standard deviation is indicated.
Name Cignal Lenti VDRE Reporter (luc) (CLS-9029L)
Official symbol TGFB1 [Human]
Official name transforming growth factor, beta 1
Species Human (Homo sapiens)
Entrez Gene ID 7040
Transcript(s) for this gene NM_000660 (2583 bp), ENST00000600196, ENST00000221930
Official symbol Tgfb1 [Mouse]
Official name transforming growth factor, beta 1
Species Mouse (Mus musculus)
Entrez Gene ID 21803
Transcript(s) for this gene NM_011577 (2191 bp), ENSMUST00000171757
Official symbol Tgfb1 [Rat]
Official name transforming growth factor, beta 1
Species Rat (Rattus norvegicus)
Entrez Gene ID 59086
Transcript(s) for this gene NM_021578 (1482 bp)
Official symbol CYP24A1 [Human]
Official name cytochrome P450, family 24, subfamily A, polypeptide 1
Species Human (Homo sapiens)
Entrez Gene ID 1591
Transcript(s) for this gene ENST00000487593, ENST00000216862, ENST00000395954, ENST00000395955, XM_005260304 (3396 bp), NM_001128915 (3089 bp), NM_000782 (3287 bp)
Official symbol Cyp24a1 [Mouse]
Official name cytochrome P450, family 24, subfamily a, polypeptide 1
Species Mouse (Mus musculus)
Entrez Gene ID 13081
Transcript(s) for this gene NM_009996 (3311 bp)
Official symbol Cyp24a1 [Rat]
Official name cytochrome P450, family 24, subfamily a, polypeptide 1
Species Rat (Rattus norvegicus)
Entrez Gene ID 25279
Transcript(s) for this gene NM_201635 (1545 bp), XM_006235672 (3209 bp)
Official symbol Vdr [Rat]
Official name vitamin D (1,25- dihydroxyvitamin D3) receptor
Species Rat (Rattus norvegicus)
Entrez Gene ID 24873
Transcript(s) for this gene NM_017058 (2043 bp)
Official symbol VDR [Human]
Official name vitamin D (1,25- dihydroxyvitamin D3) receptor
Species Human (Homo sapiens)
Entrez Gene ID 7421
Transcript(s) for this gene ENST00000229022, ENST00000395324, ENST00000549336, ENST00000550325, XM_006719587 (3474 bp), NM_000376 (4669 bp), NM_001017535 (4791 bp), NM_001017536 (5060 bp)
Official symbol Vdr [Mouse]
Official name vitamin D receptor
Species Mouse (Mus musculus)
Entrez Gene ID 22337
Transcript(s) for this gene ENSMUST00000139656, NM_009504 (4354 bp)

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