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
For Internal Use Only
-1-
Sample & Assay Technologies

2. 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
For Internal Use Only
-2-
Sample & Assay Technologies

3. 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
For Internal Use Only
-3-
Sample & Assay Technologies

4. 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.
For Internal Use Only
-4-
RT2 Profiler PCR Array
Apoptosis Pathway
Sample & Assay Technologies

5. 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
For Internal Use Only
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!!
-5-
Sample & Assay Technologies

6. 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
For Internal Use Only
-6-
Sample & Assay Technologies

7. 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?
For Internal Use Only
-7-
Sample & Assay Technologies

8. 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
For Internal Use Only
-8-
Sample & Assay Technologies

9. Cignal Lenti Reporters: Benefits
Transduce any mammalian cell
Primary cells
Stem cells
Difficult-to-transfect cell lines
.
Transduction-ready
No lentiviral generation
No titering assays
.
Versatile System
Transient experiments
Generate pathway sensor cell
lines
.
For Internal Use Only
-9-
Sample & Assay Technologies

10. 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
For Internal Use Only
- 10 -
Sample & Assay Technologies

11. 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
For Internal Use Only
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

12. Cignal Reporter Assays (45 Pathways)
Pathway
Amino Acid Deprivation
Androgen
Antioxidant Response
ATF6
C/EBP
cAMP/PKA
Cell Cycle
DNA Damage
EGR1
ER Stress
Estrogen
GATA
Glucocorticoid
Heat Shock Response
Heavy Metal Stress
Hedgehog
HNF4
Hypoxia
Interferon Regulation
Type I Interferon
Interferon Gamma
KLF4
Liver X
For Internal Use Only
Pathway
MAPK/ERK
MAPK/JNK
MEF2
c-myc
Nanog
NFkB
Notch
Oct4
Pax6
PI3K/AKT
PKC/Ca++
PPAR
Progesterone
Retinoic Acid
Retinoid X
Sox2
SP1
STAT3
TGF-beta
VDRE
Wnt
Xenobiotic
Transcription Factor
ATF4/ATF3/ATF2
Androgen Receptor
Nrf2 & Nrf1
ATF6
C/EBP
CREB
E2F/DP1
p53
EGR1
CBF/NF-Y/YY1
ER
GATA
GR
HSF
MTF1
Gli
HNF4
HIF-1
IRF-1
STAT1/STAT2
STAT1/STAT1
KLF4
LXR
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Transcription Factor
Elk-1/SRF
AP-1
MEF2
Myc/Max
Nanog
NFkB
RBP-Jk
Oct4
Pax6
FOXO
NFAT
PPAR
PR
RAR
RXR
Sox2
SP1
STAT3
SMAD2/3/4
Vitamin D Receptor
TCF/LEF
AhR
Sample & Assay Technologies

13. Cignal Reporter Assays (45 Pathways)
Pathway
Amino Acid Deprivation
Androgen
Antioxidant Response
ATF6
C/EBP
cAMP/PKA
Cell Cycle
DNA Damage
EGR1
ER Stress
Estrogen
GATA
Glucocorticoid
Heat Shock Response
Heavy Metal Stress
Hedgehog
HNF4
Hypoxia
Interferon Regulation
Type I Interferon
Interferon Gamma
KLF4
Liver X
For Internal Use Only
Transcription Factor
ATF4/ATF3/ATF2
Androgen Receptor
Nrf2 & Nrf1
ATF6
C/EBP
CREB
E2F/DP1
p53
EGR1
CBF/NF-Y/YY1
ER
GATA
GR
HSF
MTF1
GLI
HNF4
HIF-1
IRF-1
STAT1/STAT2
STAT1/STAT1
KLF4
LXR
- 13 -
Pathway
MAPK/ERK
MAPK/JNK
MEF2
c-myc
Nanog
NFκB
Notch
Oct4
Pax6
PI3K/AKT
PKC/Ca++
PPAR
Progesterone
Retinoic Acid
Retinoid X
Sox2
SP1
STAT3
TGFβ
Vitamin D
Wnt
Xenobiotic
Transcription Factor
Elk-1/SRF
AP-1
MEF2
Myc/Max
Nanog
NFκB
RBP-Jk
Oct4
Pax6
FOXO
NFAT
PPAR
PR
RAR
RXR
Sox2
SP1
STAT3
SMAD2/3/4
Vitamin D Receptor
TCF/LEF
AhR
Sample & Assay Technologies

14. Cignal Lenti Reporter Assays (35 Pathways)
Pathway
Amino Acid Deprivation
Androgen
Antioxidant Response
ATF6
C/EBP
cAMP/PKA
Cell Cycle
DNA Damage
EGR1
ER Stress
Estrogen
GATA
Glucocorticoid
Heat Shock Response
Heavy Metal Stress
Hedgehog
HNF4
Hypoxia
Interferon Regulation
Type I Interferon
Interferon Gamma
KLF4
Liver X
For Internal Use Only
Pathway
MAPK/ERK
MAPK/JNK
MEF2
c-myc
Nanog
NFkB
Notch
Oct4
Pax6
PI3K/AKT
PKC/Ca++
PPAR
Progesterone
Retinoic Acid
Retinoid X
Sox2
SP1
STAT3
TGFβ
β
VDRE
Wnt
Xenobiotic
Transcription Factor
ATF4/ATF3/ATF2
Androgen Receptor
Nrf2 & Nrf1
ATF6
C/EBP
CREB
E2F/DP1
p53
EGR1
CBF/NF-Y/YY1
ER
GATA
GR
HSF
MTF1
Gli
HNF4
HIF-1
IRF-1
STAT1/STAT2
STAT1/STAT1
KLF4
LXR
- 14 -
Transcription Factor
Elk-1/SRF
AP-1
MEF2
Myc/Max
Nanog
NFkB
RBP-Jk
Oct4
Pax6
FOXO
NFAT
PPAR
PR
RAR
RXR
Sox2
SP1
STAT3
SMAD2/3/4
Vitamin D Receptor
TCF/LEF
AhR
Sample & Assay Technologies

15. 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
For Internal Use Only
- 15 -
Sample & Assay Technologies

16. 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)
For Internal Use Only
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Sample & Assay Technologies

17. 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
For Internal Use Only
- 17 -
Renilla Luciferase
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
For Internal Use Only
- 18 -
Reporter Protein is
expressed when TF
binds to TRE
Sample & Assay Technologies

19. 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
For Internal Use Only
- 19 -
Sample & Assay Technologies

20. 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
For Internal Use Only
- 20 -
Sample & Assay Technologies

21. Cignal TRE Maximize Sensitivity and Specificity
Experimentally optimized
Cignal p53 TRE
Unoptimized p53 TRE
160
3
140
Relative Luciferase Units
Relative Luciferase Units
3.5
2.5
2
2.5-Fold
1.5
1
0.5
0
120
100
80
60
125-Fold
40
20
0
p53 Reporter
+ DMSO
For Internal Use Only
p53 Reporter
+ 1µM Doxorubicin
Cignal p53 Reporter
+ DMSO
- 21 -
Cignal p53 Reporter
+ 1µM Doxorubicin
Sample & Assay Technologies

22. 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
For Internal Use Only
- 22 -
Sample & Assay Technologies

23. Destabilized Luc: Superior Signal:Noise Ratio
Background NFkB Reporter Signal
(no treatment post-transfection)
NFkB Reporter Signal
Following TNFα Induction
α
250
0.35
Relative Luciferase Units
0.3
Fold Induction
200
150
WHY?
100
50
0.25
0.2
0.15
0.1
0.05
0
0
Negative
Control
For Internal Use Only
Stable
Luciferase
Destabilized
Luciferase
Negative
Control
- 23 -
Stable
Luciferase
Destabilized
Luciferase
Sample & Assay Technologies

24. 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.
For Internal Use Only
- 24 -
Sample & Assay Technologies

25. 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
For Internal Use Only
- 25 -
Sample & Assay Technologies

26. Cignal Lenti Reporters: How They Work
For Internal Use Only
- 26 -
Sample & Assay Technologies

27. 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
For Internal Use Only
- 27 -
Sample & Assay Technologies

28. Cignal Lenti Reporters: Primary Cells
PKC/Ca++ Signaling Pathway Studies in Primary Arteriole Smooth Muscle Cells
Cignal Lenti Reporter System Makes This Study Possible
For Internal Use Only
- 28 -
Sample & Assay Technologies

29. Cignal Lenti Reporters: Challenging Cell Lines
NFκB Signaling Pathway Studies in D1 Murine T-cell Leukemia Cell Line
κ
Cignal Lenti Reporter System Makes This Study Possible
For Internal Use Only
- 29 -
Sample & Assay Technologies

30. 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
For Internal Use Only
- 30 -
Sample & Assay Technologies

31. Cignal Lenti Reporters: How They Work
For Internal Use Only
- 31 -
Sample & Assay Technologies

32. 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
For Internal Use Only
- 32 -
Sample & Assay Technologies

33. Cignal Lenti Reporters: Sensor Cell Lines
Generation of an NFκB Signaling Pathway Stable Sensor Cell Line
κ
Cignal Lenti Reporters Enable Rapid Pathway Sensor Cell Line Generation
For Internal Use Only
- 33 -
Sample & Assay Technologies

34. 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
For Internal Use Only
- 34 -
Sample & Assay Technologies

35. 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.
For Internal Use Only
Samuel.Rulli@QIAGEN.com
- 35 -
Sample & Assay Technologies