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Transcript of Cignal lenti
Sample & Assay Technologies- 1 -For Internal Use Only
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 -For Internal Use Only
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- 3 -For Internal Use Only
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- 4 -For Internal Use Only
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- 5 -For Internal Use Only
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
Sample & Assay Technologies- 6 -For Internal Use Only
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- 7 -For Internal Use Only
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 Transfect ed• What About Primary Cells?• What About Stem Cells?• What About Cell Lines that are Difficult to Transfect?
Sample & Assay Technologies- 8 -For Internal Use Only
• 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
Sample & Assay Technologies- 9 -For Internal Use Only
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
Sample & Assay Technologies- 10 -For Internal Use Only
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- 11 -For Internal Use Only
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- 12 -For Internal Use Only
Cignal Reporter Assays (45 Pathways)
Pathway Transcription FactorAmino Acid Deprivation ATF4/ATF3/ATF2Androgen Androgen ReceptorAntioxidant Response Nrf2 & Nrf1ATF6 ATF6C/EBP C/EBPcAMP/PKA CREBCell 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- 13 -For Internal Use Only
Cignal Reporter Assays (45 Pathways)
Pathway Transcription FactorAmino Acid Deprivation ATF4/ATF3/ATF2Androgen Androgen ReceptorAntioxidant Response Nrf2 & Nrf1ATF6 ATF6C/EBP C/EBPcAMP/PKA CREBCell 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- 14 -For Internal Use Only
Cignal Lenti Reporter Assays (35 Pathways)
Pathway Transcription FactorAmino Acid Deprivation ATF4/ATF3/ATF2Androgen Androgen ReceptorAntioxidant Response Nrf2 & Nrf1ATF6 ATF6C/EBP C/EBPcAMP/PKA CREBCell 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 STAT3TGFββββ SMAD2/3/4VDRE Vitamin D ReceptorWnt TCF/LEFXenobiotic AhR
Sample & Assay Technologies- 15 -For Internal Use Only
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
Sample & Assay Technologies- 16 -For Internal Use Only
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)
Sample & Assay Technologies- 17 -For Internal Use Only
Reporter Construct GFP/firefly luciferaseTATAbox
Tandem repeats of TRE
Pathway-targeted Transcriptional Regulatory Element s (TRE), which establish the
pathway specificity of each reporter!
Cignal Reporter Assays: Complete Solution
Rigorously optimized, functionally validated, and r eady-to-use reporters
TATAboxCMVPositive Control Construct
Negative Control Construct TATAbox
Renilla LuciferaseTATAboxCMVInternal Control Construct
GFP/firefly luciferase
GFP/firefly luciferase
Sample & Assay Technologies- 18 -For Internal Use Only
Reporter Construct GFP/firefly luciferaseTATAbox
Tandem repeats of TRE
Pathway-targeted Transcriptional Regulatory Element s (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
Sample & Assay Technologies- 19 -For Internal Use Only
Reporter Construct GFP/firefly luciferaseTATAbox
Tandem repeats of TRE
Pathway-targeted Transcriptional Regulatory Element s (TRE), which establish the
pathway specificity of each reporter
Cignal Reporter Assays: Complete SolutionMultiple Reporter Formats
TF EGFP FL
Upstream SignalingEvents
Sample & Assay Technologies- 20 -For Internal Use Only
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
Sample & Assay Technologies- 21 -For Internal Use Only
Unoptimized p53 TRE
p53 Reporter+ DMSO
p53 Reporter+ 1µM Doxorubicin
Cignal TRE Maximize Sensitivity and Specificity
Experimentally optimizedCignal p53 TRE
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Cignal p53 Reporter+ DMSO
Cignal p53 Reporter+ 1µM Doxorubicin
125-Fold2.5-Fold
Sample & Assay Technologies- 22 -For Internal Use Only
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- 23 -For Internal Use Only
Destabilized Luc: Superior Signal:Noise Ratio
NFkB Reporter SignalFollowing TNF αααα Induction
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WHY?
NegativeControl
Stable Luciferase
DestabilizedLuciferase
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Background NFkB Reporter Signal(no treatment post-transfection)
NegativeControl
Stable Luciferase
DestabilizedLuciferase
Sample & Assay Technologies- 24 -For Internal Use Only
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 li nks.
Sample & Assay Technologies- 25 -For Internal Use Only
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- 27 -For Internal Use Only
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 4-8μg/ml
4) MOIs > 50 should be Split Sequential 4 hour Transductions
Sample & Assay Technologies- 28 -For Internal Use Only
Cignal Lenti Reporters: Primary Cells
PKC/Ca++ Signaling Pathway Studies in Primary Arteriole Smoo th Muscle Cells
Cignal Lenti Reporter System Makes This Study Possibl e
Sample & Assay Technologies- 29 -For Internal Use Only
Cignal Lenti Reporters: Challenging Cell Lines
NFκκκκB Signaling Pathway Studies in D1 Murine T-cell Leuke mia Cell Line
Cignal Lenti Reporter System Makes This Study Possibl e
Sample & Assay Technologies- 30 -For Internal Use Only
Cignal Lenti Reporters: Challenging Cell Lines
Cignal Lenti SRE Reporter (GFP) Measures MAPK/ERK Pat hway Signaling Activityin African Green Monkey CV1 Fibroblasts
Cignal Lenti Reporter System Makes This Study Possibl e
Sample & Assay Technologies- 32 -For Internal Use Only
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
Sample & Assay Technologies- 33 -For Internal Use Only
Cignal Lenti Reporters: Sensor Cell Lines
Generation of an NF κκκκB Signaling Pathway Stable Sensor Cell Line
Cignal Lenti Reporters Enable Rapid Pathway Sensor Ce ll Line Generation
Sample & Assay Technologies- 34 -For Internal Use Only
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
Sample & Assay Technologies- 35 -For Internal Use Only
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]