Cignal lenti webinar

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  • 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 -1- 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 -2- 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. -3- 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!! -4- 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 -5- 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? -6- 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 -7- Sample & Assay Technologies
  • 8. 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 . -8- 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 -9- 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 - 10 - 10-Pathway Arrays Cignal Lenti GFP Reporters Single Pathway Assays Sample & Assay Technologies
  • 11. 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 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 - 11 - 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
  • 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 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 - 12 - 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
  • 13. 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 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 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 - 13 - 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
  • 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 - 14 - 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) - 15 - 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 - 16 - 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 - 17 - 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 - 18 - 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 - 19 - Sample & Assay Technologies
  • 20. 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 1.5 2.5-Fold 1 0.5 0 120 100 80 60 125-Fold 40 20 0 p53 Reporter + DMSO p53 Reporter + 1µM Doxorubicin Cignal p53 Reporter + DMSO - 20 - Cignal p53 Reporter + 1µM Doxorubicin 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 - 21 - 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 - 22 - 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. - 23 - 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 - 24 - Sample & Assay Technologies
  • 25. Cignal Lenti Reporters: How They Work - 25 - 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 - 26 - 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 - 27 - Sample & Assay Technologies
  • 28. Cignal Lenti Reporters: Challenging Cell Lines NFB Signaling Pathway Studies in D1 Murine T-cell Leukemia Cell Line Cignal Lenti Reporter System Makes This Study Possible - 28 - 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 - 29 - Sample & Assay Technologies
  • 30. Cignal Lenti Reporters: How They Work - 30 - 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 - 31 - Sample & Assay Technologies
  • 32. Cignal Lenti Reporters: Sensor Cell Lines Generation of an NFB Signaling Pathway Stable Sensor Cell Line Cignal Lenti Reporters Enable Rapid Pathway Sensor Cell Line Generation - 32 - 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 - 33 - 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 - 34 - Sample & Assay Technologies