1. Cignal™ Lenti Reporter Assays
.
Pathway Reporters for Any Mammalian Cell Type
Samuel Rulli,Ph.D.
Samuel.Rulli@QIAGEN.com
Technical Scientists: 888-503-3187 or support@sabiosciences.com
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Sample & Assay Technologies
2. Topics to be Covered
Utility of the Cignal Lenti Reporters
.
• Challenges with Cell-Based Signaling Assays
• Solutions the Cignal Lenti Reporters Provide
How Cignal Lenti Reporters Work
.
• Product Breadth (multiple pathways, reporter genes, & product formats)
• Engineered for High Performance (reporter genes and TRE)
• Biosafety Features
How YOU Can Use Cignal Lenti Reporters
.
• Transient Transduction Studies in Difficult to Transfect Cell Types
• Generating Stable Pathway Sensor Cell Lines
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Sample & Assay Technologies
3. SABiosciences: Pathway Biology with a
Systems Approach
•
A leader in SYBR Green real-time PCR
technology and assays
• Focus on applying a “Pathway or Systems
Biology” approach to understanding complex
biological processes
• Sample prep to final data analysis
•
Develop gene expression-based and cell-based
assays
• PCR Arrays (Gene expression)
• Cignal™ Pathway Reporters
•
All SABiosciences products are directly available
from QIAGEN. Please contact QIAGEN or
QIAGEN distributors in your country for technical
and ordering information.
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RT2 Profiler PCR Array
Apoptosis Pathway
Sample & Assay Technologies
4. Systems Biology @ SABiosciences
Research
DNA
Genomics &
Regulation
Technologies
Sequencing
SNP Detection
ChIP
Methylation
RNA
Discovery
Gene Expression
Screening
SABiosciences Products
Illumina Service
ChIP-qPCR Assays
Methyl-Profiler
Confirmation
Protein
Gene Function
Protein Function
Gene Expression Service Core
RT2 PCR Arrays
RT2 miRNA Arrays and Assays
RT2 Individual Assays
Detection
Knockdown
Reporter System
Phenotypes
ELISArray Kits
shRNA Plasmids
Cignal Reporter System
Transcriptome PCR Arrays New!!
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Sample & Assay Technologies
5. Topics to be Covered
.
Utility of the Cignal Lenti Reporters
• Challenges with Cell-Based Signaling Assays
• Solutions the Cignal Lenti Reporters Provide
.
How Cignal Lenti Reporters Work
• Product Breadth (multiple pathways, reporter genes, & product formats)
• Engineered for High Performance (reporter genes and TRE)
• Biosafety Features
.
How YOU Can Use Cignal Lenti Reporters
• Transient Transduction Studies in Difficult to Transfect Cell Types
• Generating Stable Pathway Sensor Cell Lines
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Sample & Assay Technologies
6. Cell-Based Signaling Studies: The Challenges
• Assay Performance
• Reproducibility
• Sensitivity
• Signal-to-Noise Ratio
• Ease of Use
• Reporter Gene Limitations
• Frustration of Analyzing One Pathway at a Time
• Time Consuming
• Misleading
• Studies Limited to Cells That Are Easily Transfected
• What About Primary Cells?
• What About Stem Cells?
• What About Cell Lines that are Difficult to Transfect?
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Sample & Assay Technologies
7. Solutions Provided by Cignal Reporters
• Engineered and Formatted for Superior Performance
• Custom engineered transcriptional response elements (TRE)
• Use reporter genes with outstanding sensitivity and specificity
• Luciferase AND GFP formats are available
• Lentiviral particles are ready for transduction right out of the box
• Cignal Finder 10-Pathway Arrays
• Enable the rapid and reliable study of multiple signaling pathways
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Sample & Assay Technologies
9. Topics to be Covered
.
Utility of the Cignal Lenti Reporters
• Challenges with Cell-Based Signaling Assays
• Solutions the Cignal Lenti Reporters Provide
.
How Cignal Lenti Reporters Work
• Product Breadth (multiple pathways, reporter genes, & product formats)
• Engineered for High Performance (reporter genes and TRE)
• Biosafety Features
.
How YOU Can Use Cignal Lenti Reporters
• Transient Transduction Studies in Difficult to Transfect Cell Types
• Generating Stable Pathway Sensor Cell Lines
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Sample & Assay Technologies
10. Cignal Pathway Reporter System Products
Primary cells, stem cells,
and difficult to transfect cell lines
Easy to
transfect cell lines
Endpoint
Assays
Cignal Dual-Luciferase
Reporter Assays
Single Pathway
Assays
10-Pathway
Arrays
Dynamic
Live Cell Assays
Dynamic
Live Cell Assays
Endpoint
Assays
Cignal GFP
Reporter Assays
Cignal Lenti
Luciferase Reporters
Single Pathway
Assays
Single Pathway
Assays
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10-Pathway
Arrays
Cignal Lenti
GFP Reporters
Single Pathway
Assays
Sample & Assay Technologies
14. Cignal Reporter Experiment: Simple Workflow
What You Will Need
• Cignal Lenti Reporter or 10-Pathway Array
• SureENTRY™ can enhance transduction
efficiency in most cell types
• Test Biological
• siRNA (Flexitube siRNA)
• shRNA (SureSilencing shRNA)
• expression vectors (Qiagenes)
• protein/peptide
• small molecule
• Luciferase Assay Kit (Promega)
and luminometer or GFP detection system
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Sample & Assay Technologies
15. Cignal Lenti Reporters: Two Reporter Modalities
Dual-luciferase Format
- Quantitative endpoint luminescence assay
- Exceptional reproducibility, sensitivity, and signal-to-noise ratio
- Average expression from a population of cells
GFP Format
- Dynamic live cell assay
- Single cell resolution
- Readout flexibility
(flow cytometry, fluorescent microscopy, fluorometry)
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Sample & Assay Technologies
16. Cignal Reporter Assays: Complete Solution
Pathway-targeted Transcriptional Regulatory Elements
(TRE), which establish the
pathway specificity of each reporter!
TATA
box
Reporter Construct
GFP/firefly luciferase
Tandem repeats of
TRE
Rigorously optimized, functionally validated, and ready-to-use reporters
TATA
box
Negative Control Construct
GFP/firefly luciferase
GFP/firefly luciferase
Positive Control Construct
CMV
TATA
box
Internal Control Construct
CMV
TATA
box
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Renilla Luciferase
Sample & Assay Technologies
17. Cignal Reporter Assays: Complete Solution
Multiple Reporter Formats
Pathway-targeted Transcriptional Regulatory Elements
(TRE), which establish the
pathway specificity of each reporter
TATA
box
Reporter Construct
GFP/firefly luciferase
Tandem repeats of
TRE
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Reporter Protein is
expressed when TF
binds to TRE
Sample & Assay Technologies
18. Cignal Reporter Assays: Complete Solution
Multiple Reporter Formats
Pathway-targeted Transcriptional Regulatory Elements
(TRE), which establish the
pathway specificity of each reporter
TATA
box
Reporter Construct
GFP/firefly luciferase
Tandem repeats of
TRE
EGFP
TF
FL
Upstream Signaling
Events
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Sample & Assay Technologies
19. Cignal Reporter Optimization Process
Improved Sensitivity and Biological Relevance:
Experimentally optimized response elements :
• Sequence of transcription response element (TRE)
• Number of TREs
• Intervening sequence between TRE
Maximize specificity
and sensitivity
Engineered reporter genes:
• Destabilized firefly luciferase (Maximize signal-to-noise ratio)
• Codon optimization for mammalian expression (Maximize expression)
• Removal of hidden transcription factor binding sites (Reduce background)
Save Time and Resources: Use Cignal Reporters
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Sample & Assay Technologies
21. Cignal Reporter Optimization Process
Improved Sensitivity and Biological Relevance:
Experimentally optimized response elements :
• Sequence of transcription response element (TRE)
• Number of TREs
• Intervening sequence between TRE
Maximize specificity
and sensitivity
Engineered reporter genes:
• Destabilized firefly luciferase (Maximize signal-to-noise ratio)
• Codon optimization for mammalian expression (Maximize expression)
• Removal of hidden transcription factor binding sites (Reduce background)
Save Time, Money, and Labor: Use Cignal Reporters
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Sample & Assay Technologies
22. Destabilized Luc: Superior Signal:Noise Ratio
Background NFkB Reporter Signal
(no treatment post-transfection)
NFkB Reporter Signal
Following TNF Induction
250
0.35
0.3
Relative Luciferase Units
Fold Induction
200
150
WHY?
100
50
0.25
0.2
0.15
0.1
0.05
0
0
Negative
Control
Stable
Luciferase
Destabilized
Luciferase
Negative
Control
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Stable
Luciferase
Destabilized
Luciferase
Sample & Assay Technologies
23. Cignal Lenti Reporters: Biosafety
• VSV-G pseudotyped lentiviral particles
• Deletion in U3 portion of 3’LTR results
in self-inactivation (SIN), following
transduction of target cell
• No structural or replication genes
are expressed in transduced cells, making
Cignal lentiviral vectors
replication-defective
• No virulence genes (vpr, vif, vpu, nef) are
present in the Cignal Lenti Reporters
• Experiments should be performed under
Biosafety Level 2 (BSL-2) conditions
See our Cignal Lenti Reporter web pages for useful links.
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Sample & Assay Technologies
24. Topics to be Covered
.
Utility of the Cignal Lenti Reporters
• Challenges with Cell-Based Signaling Assays
• Solutions the Cignal Lenti Reporters Provide
.
How Cignal Lenti Reporters Work
• Product Breadth (multiple pathways, reporter genes, & product formats)
• Engineered for High Performance (reporter genes and TRE)
• Biosafety Features
.
How YOU Can Use Cignal Lenti Reporters
• Transient Transduction Studies in Difficult to Transfect Cell Types
• Generating Stable Pathway Sensor Cell Lines
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Sample & Assay Technologies
26. Tips for a Successful Transduction
1) Avoid Freeze Thaws
Result in Activity Loss
2) MOI Dilution Series
Recommended Range 5-50
3) Transduction Reagent
SureENTRY usually in the range of 4-8 μg/ml
4) MOIs > 50 should be Split
Sequential 4 hour Transductions
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Sample & Assay Technologies
27. Cignal Lenti Reporters: Primary Cells
PKC/Ca++ Signaling Pathway Studies in Primary Arteriole Smooth Muscle Cells
Cignal Lenti Reporter System Makes This Study Possible
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Sample & Assay Technologies
28. Cignal Lenti Reporters: Challenging Cell Lines
NFB Signaling Pathway Studies in D1 Murine T-cell Leukemia Cell Line
Cignal Lenti Reporter System Makes This Study Possible
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Sample & Assay Technologies
29. Cignal Lenti Reporters: Challenging Cell Lines
Cignal Lenti SRE Reporter (GFP) Measures MAPK/ERK Pathway Signaling Activity
in African Green Monkey CV1 Fibroblasts
Cignal Lenti Reporter System Makes This Study Possible
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Sample & Assay Technologies
31. Tips for Making Stable Cell Lines
1) Kill Curve for Puromycin
Don’t Guess 500–10,000 ng/ml
2) MOI for Stables
Perform a Transient Study to Find Appropriate MOI
3) Freeze Cells Down
Renewable Reagent
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Sample & Assay Technologies
32. Cignal Lenti Reporters: Sensor Cell Lines
Generation of an NFB Signaling Pathway Stable Sensor Cell Line
Cignal Lenti Reporters Enable Rapid Pathway Sensor Cell Line Generation
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Sample & Assay Technologies
33. Summary: Cignal Lenti Reporters
Breadth of Pathways and Arrays
.
• 35 Pathway-Focused Reporters
• 2 application-focused Cignal Finder Lenti 10-Pathway Arrays
Performance
.
Exceptional sensitivity, specificity, signal-to-noise ratio, and reproducibility,
using either luciferase or GFP reporter formats
High Efficiency Lentiviral Delivery System
.
• Study cell signaling in primary cells, stem cells, and difficult to
transfect cell lines
• Rapidly generate pathway sensor cell lines
Final Benefit to Researchers:
.
• Ready-to-use, validated reporters
• Don’t have to produce or amplify lentivirus in your laboratory
•Quick generation of reporter cell lines
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Sample & Assay Technologies
34. Try Cignal Reporter Assays in Your Research
Questions?
Contact Technical Support 9 AM – 6 PM Eastern M – F
Telephone: 888-503-3187;
Email: support@SABiosciences.com
Samuel J. Rulli, Jr., Ph.D.
Samuel.Rulli@QIAGEN.com
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Sample & Assay Technologies
