cignal lenti webinar

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]

mailto:[email protected]


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


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


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


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?


• 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


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


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




10-PathwayArrays

10-PathwayArrays


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


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

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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


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


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)


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





pathway specificity of each reporter

Cignal Reporter Assays: Complete SolutionMultiple Reporter Formats

Reporter Protein is expressed when TF

binds to TRE





pathway specificity of each reporter

Cignal Reporter Assays: Complete SolutionMultiple Reporter Formats

TF EGFP FL

Upstream SignalingEvents


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


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


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


Destabilized Luc: Superior Signal:Noise Ratio

NFkB Reporter SignalFollowing TNF

Induction

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WHY?

NegativeControl

Stable Luciferase

DestabilizedLuciferase

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0.25

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Background NFkB Reporter Signal(no treatment post-transfection)

NegativeControl

Stable Luciferase

DestabilizedLuciferase


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.


Cignal Lenti Reporters: How They Work


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


Cignal Lenti Reporters: Primary Cells

PKC/Ca++ Signaling Pathway Studies in Primary Arteriole Smooth Muscle Cells

Cignal Lenti Reporter System Makes This Study Possible


Cignal Lenti Reporters: Challenging Cell Lines

NFB Signaling Pathway Studies in D1 Murine T-cell Leukemia Cell Line



Cignal Lenti Reporters: Challenging Cell Lines

Cignal Lenti SRE Reporter (GFP) Measures MAPK/ERK Pathway Signaling Activityin African Green Monkey CV1 Fibroblasts



Cignal Lenti Reporters: How They Work


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


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


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


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]

mailto:[email protected]

cignal lenti webinar

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