Sample & Assay Technologies- 1 -
Cignal™ Lenti Reporter Assays
. Pathway Reporters for Any Mammalian Cell Type
Samuel Rulli,[email protected]
Technical Scientists: 888-503-3187 or [email protected]
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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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.
RT2 Profiler PCR Array
Apoptosis Pathway
Sample & Assay Technologies- 4 -
Systems Biology @ SABiosciences
DNA
RNA
Protein
SequencingSNP DetectionChIPMethylation
Illumina ServiceChIP-qPCR AssaysMethyl-Profiler
Discovery Screening
Confirmation
Gene Expression Service CoreRT2 PCR ArraysRT2 miRNA Arrays and AssaysRT2 Individual Assays
DetectionKnockdownReporter SystemPhenotypes
ELISArray KitsshRNA PlasmidsCignal Reporter SystemTranscriptome PCR Arrays New!!
Genomics &Regulation
Gene Expression
Gene FunctionProtein Function
Technologies SABiosciences ProductsResearch
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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
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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• 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
Solutions Provided by Cignal Reporters
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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
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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
Sample & Assay Technologies- 10 -
Cignal Pathway Reporter System Products
Easy totransfect cell lines
Primary cells, stem cells,and difficult to transfect cell lines
EndpointAssays
EndpointAssays
Dynamic Live Cell Assays
Dynamic Live Cell Assays
Cignal Dual-LuciferaseReporter Assays
Cignal LentiLuciferase Reporters
Cignal GFP Reporter Assays
Cignal LentiGFP Reporters
Single PathwayAssays
Single PathwayAssays
Single PathwayAssays
Single PathwayAssays
10-PathwayArrays
10-PathwayArrays
Sample & Assay Technologies- 11 -
Cignal Reporter Assays (45 Pathways)
Pathway Transcription FactorAmino Acid Deprivation ATF4/ATF3/ATF2Androgen Androgen ReceptorAntioxidant Response Nrf2 & Nrf1ATF6 ATF6C/EBP C/EBPcAMP/PKA CREB Cell Cycle E2F/DP1DNA Damage p53EGR1 EGR1ER Stress CBF/NF-Y/YY1Estrogen ERGATA GATAGlucocorticoid GRHeat Shock Response HSFHeavy Metal Stress MTF1Hedgehog GliHNF4 HNF4Hypoxia HIF-1Interferon Regulation IRF-1Type I Interferon STAT1/STAT2Interferon Gamma STAT1/STAT1KLF4 KLF4Liver X LXR
Pathway Transcription FactorMAPK/ERK Elk-1/SRFMAPK/JNK AP-1MEF2 MEF2c-myc Myc/Max Nanog NanogNFkB NFkBNotch RBP-JkOct4 Oct4Pax6 Pax6PI3K/AKT FOXOPKC/Ca++ NFATPPAR PPARProgesterone PRRetinoic Acid RARRetinoid X RXRSox2 Sox2SP1 SP1STAT3 STAT3TGF-beta SMAD2/3/4VDRE Vitamin D ReceptorWnt TCF/LEFXenobiotic AhR
Sample & Assay Technologies- 12 -
Cignal Reporter Assays (45 Pathways)
Pathway Transcription FactorAmino Acid Deprivation ATF4/ATF3/ATF2Androgen Androgen ReceptorAntioxidant Response Nrf2 & Nrf1ATF6 ATF6C/EBP C/EBPcAMP/PKA CREB Cell Cycle E2F/DP1DNA Damage p53EGR1 EGR1ER Stress CBF/NF-Y/YY1Estrogen ERGATA GATAGlucocorticoid GRHeat Shock Response HSFHeavy Metal Stress MTF1Hedgehog GLIHNF4 HNF4Hypoxia HIF-1Interferon Regulation IRF-1Type I Interferon STAT1/STAT2Interferon Gamma STAT1/STAT1KLF4 KLF4Liver X LXR
Pathway Transcription FactorMAPK/ERK Elk-1/SRFMAPK/JNK AP-1 MEF2 MEF2c-myc Myc/MaxNanog NanogNFκB NFκBNotch RBP-Jk Oct4 Oct4Pax6 Pax6PI3K/AKT FOXOPKC/Ca++ NFATPPAR PPARProgesterone PRRetinoic Acid RARRetinoid X RXRSox2 Sox2SP1 SP1STAT3 STAT3TGFβ
SMAD2/3/4 Vitamin D Vitamin D ReceptorWnt TCF/LEFXenobiotic AhR
Sample & Assay Technologies- 13 -
Cignal Lenti Reporter Assays (35 Pathways)
Pathway Transcription FactorAmino Acid Deprivation ATF4/ATF3/ATF2Androgen Androgen ReceptorAntioxidant Response Nrf2 & Nrf1ATF6 ATF6C/EBP C/EBPcAMP/PKA CREB Cell Cycle E2F/DP1DNA Damage p53 EGR1 EGR1ER Stress CBF/NF-Y/YY1Estrogen ER GATA GATAGlucocorticoid GRHeat Shock Response HSFHeavy Metal Stress MTF1Hedgehog GliHNF4 HNF4Hypoxia HIF-1Interferon Regulation IRF-1Type I Interferon STAT1/STAT2Interferon Gamma STAT1/STAT1KLF4 KLF4Liver X LXR
Pathway Transcription FactorMAPK/ERK Elk-1/SRFMAPK/JNK AP-1MEF2 MEF2c-myc Myc/MaxNanog NanogNFkB NFkBNotch RBP-JkOct4 Oct4Pax6 Pax6PI3K/AKT FOXOPKC/Ca++ NFATPPAR PPARProgesterone PRRetinoic Acid RARRetinoid X RXRSox2 Sox2SP1 SP1STAT3 STAT3TGFβ
SMAD2/3/4VDRE Vitamin D ReceptorWnt TCF/LEFXenobiotic AhR
Sample & Assay Technologies- 14 -
Cignal Reporter Experiment: Simple Workflow
• Cignal Lenti Reporter or 10-Pathway Array
• SureENTRY™ can enhance transductionefficiency 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
What You Will Need
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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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Reporter Construct GFP/firefly luciferaseTATAbox
Tandem repeats of TRE
Pathway-targeted Transcriptional Regulatory Elements (TRE), which establish the
pathway specificity of each reporter!
Cignal Reporter Assays: Complete Solution
Rigorously optimized, functionally validated, and ready-to-use reporters
TATAboxCMVPositive Control Construct
Negative Control Construct TATAbox
Renilla LuciferaseTATAboxCMVInternal Control Construct
GFP/firefly luciferase
GFP/firefly luciferase
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Reporter Construct GFP/firefly luciferaseTATAbox
Tandem repeats of TRE
Pathway-targeted Transcriptional Regulatory Elements (TRE), which establish the
pathway specificity of each reporter
Cignal Reporter Assays: Complete SolutionMultiple Reporter Formats
Reporter Protein is expressed when TF
binds to TRE
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Reporter Construct GFP/firefly luciferaseTATAbox
Tandem repeats of TRE
Pathway-targeted Transcriptional Regulatory Elements (TRE), which establish the
pathway specificity of each reporter
Cignal Reporter Assays: Complete SolutionMultiple Reporter Formats
TF EGFP FL
Upstream SignalingEvents
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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
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)
Maximize specificity and sensitivity
Save Time and Resources: Use Cignal Reporters
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Unoptimized p53 TRE
p53 Reporter+ DMSO
p53 Reporter+ 1µM Doxorubicin
Cignal TRE Maximize Sensitivity and Specificity
Experimentally optimizedCignal p53 TRE
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20
40
60
80
100
120
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160
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Cignal p53 Reporter+ DMSO
Cignal p53 Reporter+ 1µM Doxorubicin
125-Fold2.5-Fold
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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
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)
Maximize specificity and sensitivity
Save Time, Money, and Labor: Use Cignal Reporters
Sample & Assay Technologies- 22 -
Destabilized Luc: Superior Signal:Noise Ratio
NFkB Reporter SignalFollowing TNF
Induction
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50
100
150
200
250
Fold
Indu
ctio
n
WHY?
NegativeControl
Stable Luciferase
DestabilizedLuciferase
0
0.05
0.1
0.15
0.2
0.25
0.3
0.35
Rel
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Uni
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Background NFkB Reporter Signal(no treatment post-transfection)
NegativeControl
Stable Luciferase
DestabilizedLuciferase
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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 genesare expressed in transduced cells, makingCignal lentiviral vectors replication-defective
• No virulence genes (vpr, vif, vpu, nef) arepresent 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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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
Sample & Assay Technologies- 25 -
Cignal Lenti Reporters: How They Work
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Tips for a Successful Transduction
1) Avoid Freeze Thaws Result in Activity Loss
2) MOI Dilution SeriesRecommended Range 5-50
3) Transduction ReagentSureENTRY usually in the range of 4-8 μg/ml
4) MOIs > 50 should be Split Sequential 4 hour Transductions
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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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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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Cignal Lenti Reporters: Challenging Cell Lines
Cignal Lenti SRE Reporter (GFP) Measures MAPK/ERK Pathway Signaling Activityin African Green Monkey CV1 Fibroblasts
Cignal Lenti Reporter System Makes This Study Possible
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Cignal Lenti Reporters: How They Work
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Tips for Making Stable Cell Lines
1) Kill Curve for PuromycinDon’t Guess 500–10,000 ng/ml
2) MOI for StablesPerform a Transient Study to Find Appropriate MOI
3) Freeze Cells DownRenewable Reagent
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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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Summary: Cignal Lenti Reporters
. Breadth of Pathways and Arrays• 35 Pathway-Focused Reporters• 2 application-focused Cignal Finder Lenti 10-Pathway Arrays
. PerformanceExceptional 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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Try Cignal Reporter Assays in Your Research
Questions?Contact Technical Support 9 AM – 6 PM Eastern M – F
Telephone: 888-503-3187; Email: [email protected]
Samuel J. Rulli, Jr., Ph.D. [email protected]
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