Cignal Lenti Reporter User Manual
Contents
1. GFP has an excitation wavelength of 482 nm and an emission wavelength of 502 nm GFP can be detected using common fluorescence filter sets or standard FACS settings as used for EGFP and FITC Benefits of Cignal Lenti Reporter Assays Ready to transduce Delivered as transduction ready lentiviral particles eliminating any need to construct and amplify lentivirus Transduce any cell type Transduce virtually any cell type including non dividing cells stem cells and differentiated cells Wide application Can be used for transient experiments as well as for developing stable pathway sensor cell for a specific cell signaling pathway using either luciferase or GFP reporter gene technology Minimal cellular stress Lentiviral reporter construct delivery method does not produce the non specific cellular stress responses associated with chemical or electroporation based transfection methods Transient Establish Pathway Transduction Studies Sensor Cell Lines l Plate Cells Plate Cells l Cignal Lenti Reporter live lentivirus particles d Transduction Transduction l w Cignal Lenti w Cignal Lenti Antibiotic l Selection Assay Clonal Cell l Readout Line Isolation Figure 1 Overview of Cignal Lenti Pathway Reporter Applications The Cignal Lenti Reporter Assays are ready for transduction right out of the box There is no need to generate or propagate lentivirus in your laboratory These vectors are useful2. 22 25 l Introduction Lentiviral particles have been shown to be the most effective vehicle for transferring and expressing reporter constructs in almost any mammalian cell including non dividing cells primary cells stem cells differentiated cells and difficult to transfect cell lines Cignal Lenti Reporter Assays are ready to transduce replication incompetent HIV based VSV G pseudotyped lentiviral particles Cignal lentiviral reporter particles are designed for accurate sensitive and quantitative assessment of the activation of signal transduction pathways These lentiviral particles express inducible reporter constructs that encode a reporter gene under the control of a basal promoter element TATA box joined to tandem repeats of a specific transcriptional response element TRE Transcription factor activity can serve as an indicator for the intracellular status of many signal transduction pathways Our constructs are specifically engineered for measuring changes in activity both increases and decreases of these signaling pathways Each Cignal Lenti Reporter Assay is available with either luciferase or GFP as a reporter gene Cignal Lenti Reporter Assays are valuable tools for deciphering gene function as well as determining the mechanism of action of proteins peptides ligands and small molecule compounds in cells that are not amenable to transfection The Cignal Lenti firefly luciferase reporter encodes for the mammalian codo
3. Reporter Assay System Limited License The purchaser of this product agrees to only use the lentiviral particles in this kit in cell based reporter assays for in vivo and in vitro internal research Use of this product for Commercial Purposes requires a license from Sigma Aldrich Corporation The purchase of this product conveys to the buyer the nontransferable right to use the purchased amount of the product and components of the product in research conducted by the buyer whether the buyer is an academic or for profit entity The buyer cannot sell or otherwise transfer a this product b its components or c materials made using this product or its components to a third party or otherwise use this product or its components or materials made using this product or its components for Commercial Purposes Commercial Purposes means any activity by a party for consideration but excludes not for profit core facilities providing services within their own research institutions at cost This product is licensed under U S Pat Nos 5 817 491 5 591 624 5 716 832 6 312 682 6 669 936 6 235 522 6 924 123 and foreign equivalents from Oxford BioMedica UK Ltd Oxford UK and is provided for use in academic and commercial in vitro and in vivo research for elucidating gene function and for validating potential gene products and pathways for drug discovery and development but excludes any use of LentiVector technology for creating transgenic birds for the p
4. a hemacytometer 2 Add 100 ul of resuspended cells 0 5 1x10 cells in each well of 96 well plate Triplicate wells for each lentiviral reporter negative control and positive control should be used 3 Incubate cells at 37 C overnight in a humidified 5 CO incubator Note While determining the plating density please consider that the growth rates of cells vary greatly and account for the length of time the cells will be growing before the assay development Day 2 4 Remove medium from wells To each well add 20 ul of Cignal lentiviral particles Lenti reporter or Lenti negative control or Lenti positive control and make up the total volume of 50 ul using growth medium without antibiotics DMEM with 10 FBS 0 1mM NEAA 1mM Sodium pyruvate In this particular case add 30 ul of growth medium without antibiotics 5 Add SureENTRY Transduction Reagent to a final concentration of 8 ug ml in each well Gently swirl the plate to mix 6 Incubate 18 20 hours at 37 C in a humidified incubator in an atmosphere of 5 CO Note SureENTRY Transduction Reagent enhances transduction of most cells however some cells like primary neurons are sensitive to SureENTRY Transduction Reagent Do not add SureENTRY Transduction Reagent to these types of cells If working with a cell 17 type for the first time a SureENTRY Transduction Reagent control only well should be used to determine cell sensitivity Important When transducing Cignal l
5. containing medium every 3 4 days until resistant colonies can be identified after selection Pick a minimum of 5 puromycin resistant colonies and expand each clone Check each clone for its ability to sense the modulation of the activity of the specific transcription factor or signaling pathway Use the most responsive clone for further studies To determine the appropriate amount of puromycin for selection of transduced target cells perform the puromycin titration kill curve using the following guidelines i Plate 1 6 x10 cells into wells of a 96 well plate with 120 ul fresh media ii The next day add 500 10 000 ng ml of puromycin to selected wells iii Examine viability every 2 days iv Culture for 10 14 days Replace the media containing puromycin every 3 days v The minimum concentration of puromycin that causes complete cell death after 3 5 days should be used for that cell type VI Frequently Asked Questions Q How many cells can transduce with the amount of Cignal Lenti Reporter Assays provided A The amount of cells that can be transduced depends upon your chosen target cells and how easily they are transduced Primary or other difficult cells may require higher MOls than cell lines It is recommended to perform a limiting dilution titer on your target cells utilizing our Cignal Lenti positive control cat CLS PCG to determine the optimal amount of viral particles needed for your particular cell type Q Ho
6. e Wear double gloves and lab coat at all times e Perform work in a Class Il Biosafety Cabinet BSC and post biohazard warning signs e Minimize splashes or aerosols with careful pipeting e Autoclave all biological wastes and decontaminate before disposal Ill Cignal Lenti Reporter Controls Variant ID Concentration Control for Catalog Description And Volume No 336891 Positive Control GFP CLS PCG Easily measure transduction efficiency and optimize 2 0 8 x10 TU ml transduction conditions with 250 ul per tube Green Fluorescent Protein Positive Control RFP CLS PCR Easily measure transduction efficiency and optimize 2 0 8 x10 TU ml transduction conditions with 250 ul per tube Red Fluorescent Protein New Negative Control GFP CLS NCG Establish the specificity of any treatment effects and 2 0 8 x10 TU ml determine background GFP 250 Jl per tube fluorescence Negative Control Firefly Luciferase CLS NCL Establish the specificity of any treatment effects and 2 0 8 x10 TU ml determine background firefly 250 ul per tube luciferase activity CMV Renilla Control CLS RCL Serves as an internal control for normalization in dual 2 0 8 x10 TU ml luciferase assay format 250 ul per tube providing more accurate interpretation of results TK Renilla Control luc CLS TKL Serves as an internal control 2 0 8 x10 TU ml for normalization in dual 250 ul per tube luciferase assay format providing mor
7. for transient transduction studies in difficult to transfect cells or for pathway sensor cell line generation Il Product Contents and Descriptions A Contents Table 1 Cignal Lenti Reporter Product Specifications Component Specification Lentivirus Concentration total volume Ready to transduce Lenti Reporter 1 tube HenseripHon factor gt 0 8 x10 TU ml 250 ul responsive lentiviral reporter Ready to transduce Lenti Reporter 8 tubes iransenpian factor gt 0 8 x10 TU ml 2000 ul responsive lentiviral reporter Note The exact titer of each Cignal Lentivirus preparation is reported on the Certificate of Analysis Important We recommend the use of Cignal Lenti Negative Control and Cignal Lenti Positive Control along with Cignal Lenti Reporter for better interpretation of results for more details about Cignal Lenti Controls see page 11 B Description Cignal Lenti Reporter Assay Cignal Lenti Reporter Assays are delivered as ready to transduce lentiviral particles expressing a transcription factor responsive reporter gene firefly luciferase or GFP under the control of a basal promoter element TATA box joined to tandem repeats of a specific Transcriptional Response Element TRE A schematic of the transfer vector used to generate the Cignal Lentiviruses is shown in Figure 2 Cignal Lenti Reporter Assays monitor both increases and decreases in the activity of a key transcription fa
8. test SIoRNA ShRNA plasmids or expression vectors Day 4 or 5 Depending upon experimental design treat with test proteins peptides or compounds Day 5or6 Analyze pathway reporter gene expression luciferase or GFP We recommend using the Cignal Lenti Positive Control cat no CLS PCG in an initial experiment to determine the optimal MOI for the target cells being studied SureENTRY Transduction Reagent enhances lentiviral transduction efficiency in most cell types C Detailed Protocol The following protocol is designed to transduce HEK293 cells using Cignal Lenti Reporter Assays in a 96 well plate format Cignal Lenti Reporters Assays work well with other mammalian cells If you are using plates or wells of different size adjust the components in proportion to the surface area This is just a general guideline the optimal transduction conditions should be optimized according to the cell type and the study requirements Read the protocol completely before starting the experiment Day 1 1 Trypsinize 90 confluent HEK293 cells with trypsin EDTA for 2 5 minutes at 37 C to make cell suspension Gently detach the cells from tissue culture dish with a pipette mix with one volume of culture medium containing 10 fetal bovine serum then centrifuge down remove the supernatant and suspend cells to 0 5 1 x 10 cells ml in growth media To ensure reproducible transduction results it is important to determine the cell density with
9. 1G MEF2 MEF2 CLS 4024L c myc Myc Max CLS 012L Nanog Nanog CLS 4037L NFkB NFkB CLS 013L CLS 013G Notch RBP Jk CLS 014L CLS 014G Oct4 Oct4 CLS 7025L PI3K AKT FOXO CLS 8022L PKC Ca NFAT CLS 015L Retinoic Acid Receptor Retinoic Acid Receptor RAR CLS 016L Retinoid X Receptor RXR CLS 6044L SP1 SP1 CLS 3027L STAT3 STAT3 CLS 6028L TGFB SMAD2 SMAD3 SMAD4 CLS 017L Vitamin D VDR CLS 9029L Wnt TCF LEF CLS 018L CLS 018G Xenobiotic AhR CLS 9045L 23 Cignal Lenti Reporter Controls Variant ID for Control Catalog No Description 336891 Positive Control GFP CLS PCG Easily measure transduction efficiency and optimize transduction conditions with Green Fluorescent Protein Positive Control RFP CLS PCR Easily measure transduction efficiency and optimize New transduction conditions with Red Fluorescent Protein Negative Control GFP CLS NCG Establish the specificity of any treatment effects and determine background GFP fluorescence Negative Control Firefly Luciferase CLS NCL Establish the specificity of any treatment effects and determine background firefly luciferase activity CMV Renilla Control CLS RCL Serves as an internal control for normalization in dual luciferase assay format providing more accurate interpretation of results TK Renilla Control luc CLS TKL Serves as an internal control for normalization in dual luciferase assay format providing more accurate interpretation of results CMV Ren
10. 28 10 19 Technical 0800 28 10 11 Belgium Orders 0800 79612 Fax 0800 79611 Technical 0800 79556 Brazil Orders 0800 557779 Fax 55 11 5079 4001 Technical 0800 557779 Canada Orders 800 572 9613 Fax 800 713 5951 Technical 800 DNA PREP 800 362 7737 China Orders 86 21 3865 3865 Fax 86 21 3865 3965 Technical 800 988 0325 Denmark Orders 80 885945 Fax 80 885944 Technical 80 885942 Finland Orders 0800 914416 Fax 0800 914415 Technical 0800 914413 France Orders 01 60 920 926 Fax 01 60 920 925 Technical 01 60 920 930 Offers 01 60 920 928 Germany Orders 02103 29 12000 Fax 02103 29 22000 Technical 02103 29 12400 Hong Kong Orders 800 933 965 Fax 800 930 439 Technical 800 930 425 Ireland Orders 1800 555 049 Fax 1800 555 048 Technical 1800 555 061 Italy Orders 800 789 544 Fax 02 334304 826 Technical 800 787980 Japan Telephone 03 6890 7300 Fax 03 5547 0818 Technical 03 6890 7300 Korea South Orders 080 000 7146 Fax 02 2626 5703 Technical 080 000 7145 Luxembourg Orders 8002 2076 Fax 8002 2073 Technical 8002 2067 Mexico Orders 01 800 7742 639 Fax 01 800 1122 330 Technical 01 800 7742 436 The Netherlands Orders 0800 0229592 Fax 0800 0229593 Technical 0800 0229602 Norway Orders 800 18859 Fax 800 18817 Technical 800 18712 Singapore Orders 1800 742 4362 Fax 65 6854 8184 Technical 1800 742 4368 Spain Orders 91 630 7050 Fax 91 630 5145 Technical 91 630 7050
11. August 2012 Cignal Lenti Reporter Handbook For lentiviral based cell signaling activity assays QIAGEN Sample amp Assay Technologies QIAGEN Sample and Assay Technologies QIAGEN is the leading provider of innovative sample and assay technologies enabling the isolation and detection of contents of any biological sample Our advanced high quality products and services ensure success from sample to result QIAGEN sets standards in Purification of DNA RNA and proteins Nucleic acid and protein assays microRNA research and RNAi Automation of sample and assay technologies Our mission is to enable you to achieve outstanding success and breakthroughs For more information visit www qiagen com Product Use Limitations Cignal Lenti Reporter Assays are intended for molecular biology applications These products are not intended for the diagnosis prevention or treatment of a disease CONTENTS l Introduction Il Product Contents and Descriptions A Contents B Description C Production and Titration of Cignal Lentiviral Particles D Biosafety Features of Cignal Lentiviral Particles E Safety Guidelines Il Cignal Lenti Reporter Controls IV Additional Materials Required V Protocol A Before You Begin B Brief Protocol C Detailed Protocol VI Frequently Asked Questions Appendix Troubleshooting and Cignal Lenti Reporter Products Ordering information 10 10 11 13 14 14 16 17 20
12. Number of target cells present in that well Total transducing units needed per well TU Total number of cells per well x Desired MOI Total mL of lentiviral particles to add to each well Total TU needed per well TU mL reported on Certificate of Analysis We have found that some commonly used cancer cell lines e g HT 1080 HEK293 and HepG2 can be effectively transduced by lentivectors using 10 to 25 MOI however some cell types like primary cells are more resistant to transduction and efficient transduction of these cell types may require a higher MOI around 50 Importantly it has been reported in the literature that the VSV G pseudotyped lentiviruses can be used to transduce stem cells primary cells HUVEC keratinocytes bone marrow adipose and many other cell types including neurons endothelial retinal pancreatic skin fibroblasts macrophages etc Concentration of SureENTRY Transduction Reagent SureENTRY Transduction Reagent is a small positively charged molecule that binds to cell surfaces neutralizes surface charge increases binding between pseudoviral capsid and the cellular membrane and greatly enhances transduction efficiency The optimal concentration of SureENTRY Transduction Reagent depends on cell type and may need to be determined experimentally usually in the range of 4 8 ug ml SureENTRY Transduction Reagent can be toxic to terminally differentiated neurons and dendritic cells In situations lik
13. Please follow CDC guidelines We typically use 1096 bleach to inactivate the virus Q What does transduction unit TU mean 20 A Transducing Units refer to the number of vector genomes that can infect enter and integrate into a population of cells Q What precautions one should take while handling Cignal Lentiviruses A The Cignal Lentiviruses should be used in a BSL2 tissue culture cabinet using gloves and BSL2 tissue culture procedures For any other troubleshooting or technical questions about the Cignal Lenti Reporters please call one of our Technical Support representatives at 1 888 503 3187 or 301 682 9200 or email at support SABiosciences com 21 Appendix Troubleshooting and Cignal Lenti Reporter Products A Reasons for inefficient transduction or low expression of Cignal Lenti Reporters Target cell type may be difficult to transduce Optimize the transduction protocol number of cell SureENTRY Transduction Reagent concentration and MOI required for best transduction and use higher MOI In some cases SureENTRY Transduction Reagent is toxic for target cells Volume of Cignal lentiviral particles used is too high Keep the volume as low as possible to achieve maximal adsorption of viral particles to the cells The assay is performed too early Usually the maximal expression of integrated transgene is expected to develop by 72 hours after infection however some cells showed delayed expression Therefore we sug
14. Sweden Orders 020 790282 Fax 020 790582 Technical 020 798328 Switzerland Orders 055 254 22 11 Fax 055 254 22 13 Technical 055 254 22 12 e e UK Orders 01293 422 911 Fax 01293 422 922 Technical 01293 422 999 oo0000 USA Orders 800 426 8157 Fax 800 718 2056 Technical 800 DNA PREP 800 362 7737 QIAGEN eee Sample amp Assay Technologies
15. ctor which is a downstream target of a specific signaling pathway Reporter gene cppt TATA box PGK PuroR RRE SIN 3 LTR pCignal Lenti TRE Reporter gene Psi RSV 5 LTR gt f1 ori AmpR Figure 2 Schematic of lentiviral vector used to generate Cignal Lenti Reporter Assays pCignal Lenti TRE Reporter gene encodes the inducible transcription factor responsive construct expressing firefly luciferase or GFP as a reporter gene The important features of the vector are described in the table below Feature Function RSV 5 LTR Hybrid Rous sarcoma Virus Permits viral packaging and reverse RSV enhancer promoter U5 long terminal transcription of viral MRNA repeat Psi Packaging signal Allow viral packaging RRE Rev response element Involved in packaging of viral transcript cppt Central polypurine tract Involved in nuclear translocation and integration of transduced viral genome Reporter gene firefly luciferase or GFP Allow quantification of transcription hPGK human phosphoglycerate kinase Permits high level expression of the eukaryotic promoter mammalian selection marker puromycin PuroR puromycin resistance gene Can be used for mammalian selection SIN 3 LTR 3 self inactivating long terminal Modified 3 LTR that allows viral packaging repeat but self inactivates the 5 LTR for biosafety purpose The element also contains a polyadenylation signal for efficient transcripti
16. determinations of luminescence activity resulting from the research use of this product and its derivatives or 2 contact Promega Corporation to obtain a license for the use of the product and its derivatives No other use or transfer of this product or its derivatives is authorized without the express written consent of Promega Corporation including without limitation Commercial Use Commercial Use means any and all uses of this product and derivatives by a party for monetary or other consideration and may include but is not limited to use in 1 product manufacture and 2 to provide a service information or data and or resale of the product or its derivatives whether or not such product or derivatives are resold for use in research With respect to such Commercial Use or any diagnostic therapeutic or prophylactic uses please contact Promega Corporation for supply and licensing information If the purchaser is not willing to accept the conditions of this limited use statement SABiosciences is willing to accept the return of the unopened product and provide the purchaser with a full refund However in the event the product is opened then the purchaser agrees to be bound by the conditions of this limited use statement The above license relates to Promega Corporation patents and or patent applications on improvements to the luciferase and Monster Green gene United States Patent No 5 292 658 licensed from Millipore Corporation Cignal Lenti
17. e accurate interpretation of results New CMV Renilla Control Hygromycin CLS RHL For generating stable cell 2 0 8 x10 TU ml lines this construct serves as 250 ul per tube an internal control for normalization in dual luciferase assay format New Positive Control luc CLS PCL Measure transduction 2 0 8 x10 TU ml efficiency and serve as 250 ul per tube positive control for firefly luciferase assay ml ml ml ml u u M Description of Cignal Lenti Reporter Controls 1 Cignal Lenti Negative control The Cignal Lenti negative controls are ready to transduce lentiviral particles expressing firefly luciferase or GFP under the control of a basal promoter element TATA box without any additional transcriptional response elements Figure 3a The negative control is critical to establishing the specificity of any treatment effects and determining background reporter activity 11 2 Cignal Lenti Positive control The Cignal Lenti positive controls are ready to transduce lentiviral particles constitutively expressing either firefly luciferase or GFP Figure 3b The Cignal Lenti positive control GFP is necessary for visual confirmation of transduction It is also useful for transduction optimization studies GFP has an excitation wavelength of 482 nm and an emission wavelength of 502 nm GFP can be detected using common fluorescence filter sets or standard FACS settings as used for EGFP and FITC 3 Cignal Le
18. e this titration of SureENTRY Transduction Reagent using 2 4 6 8 ug ml will have to be done to determine the highest nontoxic concentration that can be used If toxicity is a big problem then cells can be transduced in the absence of SureENTRY Transduction Reagent but the MOI will have to be increased Time of assay development lt is recommended to wait a minimum of 48 hours after lentiviral transduction to allow the reporter gene present in the lentiviral vector to reverse transcribe and integrate into the chromosomal DNA In most cases expression of reporter gene can be measured 72 hours after transduction transient transduction However some cell types show a delay in expressing reporter genes In these cases we recommend development of reporter assay at about 96 hours after transduction Transient transduction or stable cell generation Cignal Lenti Reporter Assays work very well for transient transduction experiments In such transient pathway activation studies reporter gene expression is typicaly measured 72 to 96 hours after transduction At that time Cignal reporter constructs are integrated into the genomic DNA These cells can be further cultured under puromycin selection to generate stably transduced signaling pathway sensor cell lines Some cells lines like primary cell lines only express the Cignal reporter construct in 10 3096 of cells even when transduced at high MOl s For these difficult to transduce cells it is impor
19. echnical Services or your local distributor 25 26 Trademarks QIAGEN QIAGEN Group Limited License Agreement Use of this product signifies the agreement of any purchaser or user of Cignal Lenti Reporter Assays to the following terms 1 The Cignal Lenti Reporter Assays may be used solely in accordance with the Cignal Lenti Reporter Handbook and for use with components contained in the Kit only QIAGEN grants no license under any of its intellectual property to use or incorporate the enclosed components of this Kit with any components not included within this Kit except as described in the Cignal Lenti Reporter Handbook and additional protocols available at www qiagen com Other than expressly stated licenses QIAGEN makes no warranty that this Kit and or its use s do not infringe the rights of third parties This Kit and its components are licensed for one time use and may not be reused refurbished or resold QIAGEN specifically disclaims any other licenses expressed or implied other than those expressly stated U BO NS The purchaser and user of the Kit agree not to take or permit anyone else to take any steps that could lead to or facilitate any acts prohibited above QIAGEN may enforce the prohibitions of this Limited License Agreement in any Court and shall recover all its investigative and Court costs including attorney fees in any action to enforce this Limited License Agreement or any of its intellectual property rights re
20. entiviral particles into a cell type for the first time we suggest using either 10 or 50 MOI as a starting point to determine the optimal assay development conditions Always include Cignal Lenti positive control GFP for determining transduction efficiency Day 3 7 Remove the medium containing Cignal lentiviral particles from wells Add 100 ul of fresh growth medium DMEM with 10 FBS 0 1mM NEAA 1mM Sodium pyruvate 100 U ml penicillin and 100 ug ml streptomycin to each well Day 5 8 Harvest the transduced cells and assay for the expression of the reporter gene Important notes 1 In most cell types the expression of reporter gene can be measured directly at about 72 hours after transduction day 5 of the assay However some cell types show a delay in expressing reporter genes and in these cases we recommend development of reporter assay at about 96 hours after transduction day 6 of the assay 2 The luciferase assay can be developed by using either the Firefly Luciferase or Dual Luciferase Reporter Assay Systems from Promega Follow the manufacturer s protocol for developing the assay 3 The expression of the GFP reporter can be monitored via FACS flow cytometry fluorescent microscopy or standard fluorometry GFP has an excitation wavelength of 482 nm and an emission wavelength of 502 nm GFP can be detected using common fluorescence filter sets or standard FACS settings as used for EGFP and FITC 4 To determine t
21. gest developing the assay at a later time such as 96 hours Inactivation of Cignal Lentiviral particles during storage Store lentiviruses at 80 C and avoid freeze thaw cycle Cignal Lentiviral stock medium affects target cell growth Dilute the stock medium or concentrate the pseudovirus by centrifugation to minimize the amount of stock medium added to the target cells B Reasons for no expression from Cignal Lenti positive control It might be any one of the reasons stated above OR CMV promoter is not functional in target cells In certain cell types the CMV promoter is not functional In these cases one has to change the type of target cells or use lentivirus having a constitutively active promoter other than the CMV promoter 22 Ci nal Lenti Reporter Assays Pathway Transcription Factor luciferase GFP Variant ID Variant ID Amino Acid Deprivation New ATFA ATF3 ATF2 CLS 5034L Androgen AR CLS 8019L Antioxidant Response Nrf2 amp Nrf1 CLS 2020L ATF6 New ATF6 CLS 6031L C EBP C EBP CLS 001L cAMP PKA CREB CLS 002L CLS 002G Cell Cycle E2F DP1 CLS 003L EGR1 EGR1 CLS 5021L ER Stress CBF NF Y YY1 CLS 9032L Heavy Metal Stress MTF1 CLS 2033L Hedgehog GLI CLS 3030L Hypoxia HIF 1 CLS 007L Interferon Regulation IRF1 CLS 4040L Type Interferon STAT1 STAT2 CLS 008L Interferon Gamma STAT1 STAT1 CLS 009L KLF4 KLF4 CLS 1036L Liver X Receptor LXRa CLS 7041L MAPK ERK Elk 1 SRF CLS 010L CLS 010G MAPK JNK AP 1 CLS 011L CLS 01
22. he effect_of siRNA shRNA on a specific reporter or signaling pathway we recommend doing transient transfection of siRNA shRNA on day 3 of the assay 5 To determine the effect of overexpression of a gene on a specific reporter or signaling pathway we recommend doing the transient transfection of 100 200 ng of experimental vector and negative control vector 24 or 36 hours before the assay development 6 To determine the effect of recombinant protein or small peptide on a specific reporter or signaling pathway we recommend changing the cell medium to assay medium Opti MEM containing 0 5 of fetal bovine serum 1 NEAA 100 U ml 18 Penicillin and 100 ug ml Streptomycin and treating the transduced cells with 3 to 4 different concentration of recombinant protein or small peptide about 6 or 24 hours before the assay development To determine the effect of small chemicals on a specific reporter or signaling pathway we recommend changing the cell medium to assay medium Opti MEM containing 0 5 of fetal bovine serum 1 NEAA 100 U ml Penicillin and 100 ug ml Streptomycin and treating the transduced cells with 3 to 4 different concentration of small chemicals for 6 to 24 hours before the assay development For the generation of stable cell line on DAY 4 remove the growth medium and replace it with fresh growth medium that contains the appropriate amount of puromycin for selection of transduced cells Replace medium with fresh puromycin
23. illa Control Hygromycin CLS RHL For generating stable cell lines this construct serves as an internal control for normalization in dual luciferase assay format Positive Control luc CLS PCL Measure transduction efficiency and serve as positive control for firefly luciferase assay Cignal Finder Lenti 10 Pathway Reporter Arrays Product Name Components Concentration and Volume Catalog Number Cignal Finder Lenti Immune Response 10 Pathway Reporter Array Ready to transduce transcription factor responsive lentiviral firefly luciferase pathway reporters 10 tubes total negative and positive controls 1 tube each gt 0 8 x10 TU ml 250 ul of each reporter and control CLA 002L 336831 Cignal Finder Lenti Development 10 Pathway Reporter Array Ready to transduce transcription factor responsive lentiviral firefly luciferase pathway reporters 10 tubes total negative and positive controls 1 tube each gt 0 8 x10 TU ml 250 ul of each reporter and control CLA 003L 336831 24 Ordering Information Product Contents Cat no Cignal Lenti Reporter Ready to transduce transcription factor Varies Assays responsive lentiviral reporters in tube format For up to date licensing information and product specific disclaimers see the respective QIAGEN kit handbook or user manual QIAGEN kit handbooks and user manuals are available at www giagen com or can be requested trom QIAGEN T
24. lating to the Kit and or its components For updated license terms see www giagen com Firefly and or Renilla Luciferase and Monster Green Limited Use Label License READ THIS FIRST BEFORE OPENING PRODUCT For research use only The terms of the limited license conveyed with the purchase of this product are as follows Researchers may use this product in their own research and they may transfer derivatives to others for such research use provided that at the time of transfer a copy of this label license is given to the recipients and the recipients agree to be bound by the conditions of this label license Researchers shall have no right to modify or otherwise create variations of the nucleotide sequence of the luciferase gene or Monster Green gene except that Researchers may 1 clone heterologous DNA sequences at either or both ends of said luciferase or Monster Green gene so as to create fused gene sequences provided that the coding sequence of the resulting luciferase or Monster Green gene has no more than four deoxynucleotides missing at the affected terminus when compared to the intact luciferase or Monster Green gene sequence and 2 insert and remove nucleic acid sequences in furtherance of splicing research predicated on the inactivation or reconstitution of the luminescent activity of the encoded luciferase In addition Researchers must do one of the following 1 use luminescent assay reagents purchased from Promega Corporation for all
25. n optimized non secreted form of the firefly luciferase gene carrying a protein destabilizing sequence Cells rapidly degrade the destabilized form of the firefly luciferase protein and hence the background luciferase activity noise level is greatly reduced Due to low background activity the magnitude of the response that can be measured signal to noise ratio as well as the speed of measuring changes in transcription are enhanced The Cignal Lenti luciferase reporter assays provide outstanding reproducibility sensitivity specificity and signal to noise ratio They are extremely useful for carrying out endpoint pathway regulation assays The Cignal Lenti GFP Reporter assay enables you to monitor the dynamics of pathway activation on living cells with single cell resolution The Cignal Lenti GFP reporter constructs utilizes an improved version of the green fluorescent protein gene This GFP expression cassette has been codon optimized to maximize mammalian cell expression and also utilizes an optimized Kozak sequence to increase translation efficiency The synthetic GFP is an ideal fluorescent reporter providing high level fluorescence and minimal cytotoxicity Moreover the synthetic GFP gene is resistant to photobleaching In addition most consensus sequences for transcription factor binding have been removed from the synthetic GFP gene in order to minimize aberrant transcription and improve the reliability of the GFP as an accurate reporter
26. nt transduction Optimization of conditions for efficient transduction The sensitivity of the Cignal Lenti Reporter Assay depends on the transduction efficiency The transduction efficiency in turn primarily depends upon the cell type being transduced Therefore it is very important to optimize the transduction conditions for each cell type under study Variables to consider when optimizing the transduction conditions include Multiplicity of Infection MOI concentration of SureENTRY Transduction Reagent used time of assay development and the cell density The Cignal Lenti positive control GFP Cat CLS PCG can be used for determining the optimal transduction conditions Multiplicity of Infection MOI The transduction efficiency of Cignal Lenti reporters varies significantly for different cell type It is important to determine the Multiplicity of Infection MOI which is the number of transducing lentiviral particles per cell required to get desired transduction efficiency for each new cell type The MOI is typically adjusted by increasing or decreasing the amount of virus added per well to a series of wells containing the same number of cells We recommend testing the Cignal Lenti Positive Control CLS PCG at an MOI of 5 10 and 50 each MOI in triplicate in order to establish the optimal MOI for each cell type to be studied To calculate Multiplicity of Infection MOI Number of transducing units TU deposited in a well
27. nti Renilla control Rluc The Cignal Lenti Renilla control is a preparation of ready to transduce lentiviral particles constitutively expressing Renilla luciferase Figure 3b The Cignal Lenti Renilla control Rluc serves as an internal control when performing dual luciferase reporter assays The Cignal Lenti Renilla control can be helpful in overcoming technical variability and obtain more reliable data cppt n Reporter gene TATA box hPGK v PuroR RRE SIN 3 LTR pCignal Lenti minP Reporter gene Psi RSV 5 LTR f1 ori AmpR Reporter gene CPP M Gk Hc PuroR CMV SIN 3 LTR pCignal Lenti CMV Reporter gene f1 ori Psi RSV S LTR AmpR b Figure 3 Schematic of lentiviral vector used to generate Cignal Lenti Reporter controls a pCignal Lenti minP Reporter gene contains a non inducible firefly luciferase or GFP expression cassette b pCignal Lenti CMV Reporter gene contains a constitutive firefly luciferase or GFP or Renilla luciferase expression cassette The important features of the vectors are the same as those described on page 9 for the pCignal Lenti TRE Reporter gene vectors IV Additional Materials Required Mammalian cells cultured in the appropriate growth medium Cell culture medium and cell culture supplies Biosafety Level 2 BSL 2 equipment and work environment 96 well tissue culture plates Multi channel pipettor and pipettor reservoirs Hemacytometer SureENTRY Transd
28. on termination f1 ori f1 origin of replication Origin of DNA replication for bacteriophage fi AmpR ampicillin resistance gene Allows selection of the plasmid in E coli TRE Transcription response element Permits regulation of reporter gene expression by a specific transcription factor TATA box Act as an minimal promoter C Production and Titration of Cignal Lentiviral Particles The infectious replication incompetent pseudotyped Cignal Lentiviral particles were produced by cotransfecting specific Cignal Lentivector Fig 2 along with plasmids expressing packaging proteins using VSV G as an envelope protein into HEK293T cells Following cotransfection we collected the media containing the pseudoviral particles was collected centrifuged at 1250 rpm for 5 minutes and filtered through 0 45 um filter The resultant lentiviruses were aliquoted and stored at 80 C The lentiviral particles were titered by determining the number of antibiotic resistant cells colonies that arise after transduction and puromycin selection of HT 1080 cells The exact titer of Cignal Lentiviral particles reporter negative and positive controls will vary for different lots and are provided on the certificate of analysis included in each shipment D Biosafety Features of Cignal Lentiviral Particles The Cignal Lentiviral particles have numerous biosafety features which include e A deletion in the promoter enhancer region of the U3 po
29. rtion of 3 LTR ensures self inactivation of the lentiviral construct after transduction and integration into genomic DNA of target cells e The Cignal Lentivector and plasmids expressing packaging proteins contain no significant areas of homology and thereby minimizing their chance for recombination e None of the HIV 1 genes gag pol rev will be expressed in transduced cells as they are expressed from packaging plasmids lacking packaging signal Therefore the lentiviral particles that are generated are replication incompetent e No virulence genes Avpr vif vpu and nef are present in the Cignal Lentivector so Lentiviral particles will carry only a copy of reporter gene of interest E Safety Guidelines Although the Cignal lentiviral particles are replication incompetent it is highly recommended that they be treated as Risk Group Level 2 RGL 2 organisms Follow all published RGL 2 guidelines for handling and waste decontamination Details on requirements for creating a BSL 2 work environment are available in the U S Department of Health and Human Services publication Biosafety in Microbiological amp Biomedical Laboratories 4th 5th edition http www cdc gov biosafety publications bmbl5 You should also consult the health and safety guidelines and officers at your institution regarding use and handling of the lentiviral system While working with Cignal Lenti Reporters we also recommend following standard safety practices
30. tant to select the cells stably expressing the reporter gene by puromycin selection for an additional two weeks prior to carrying out pathway activation studies 3 Optimization of assay condition The response rate in the Cignal Lenti Reporter Assay depends on the assay conditions conditions of the experimental treatment To obtain maximum response given by any stimulus perform dosing and time course studies The optimal amount of stimulus and the time of treatment must be obtained empirically for each experiment see different protocols for our recommendations 4 Important recommendations for best results 1 Perform all transduction in triplicate to minimize variability among treatment groups 2 Include positive and negative controls in each experiment to obtain reliable results 15 3 Take care to always seed the same number of cells in each well in order to maximize the reproducibility of your experiment 4 Serum induces various signaling pathways leading to cross talk and high background Therefore use reduced amounts of serum 0 596 in the assay medium during the experimental treatment to minimize these serum effects B Brief Protocol Day 1 Seed cells Day 2 Remove growth medium and add appropriate amount of Cignal Lenti Reporter typically 10 to 50 MOI and SureENTRY Transduction Reagent Day 3 Remove Cignal Lenti Reporter suspension Replace with growth medium Depending upon experimental design transfect with
31. uction Reagent Reagent Catalog Number 336921 Cignal Lenti Negative Control o For firefly luciferase reporter studies cat CLS NCL o For GFP reporter studies cat CLS NCG Cignal Lenti Positive Control o For firefly luciferase reporter studies CLS PCL o For GFP reporter studies CLS PCG Cignal Lenti Renilla Control For dual luciferase reporter assay format cat CLS RCL or CLS TKL or CLS RHL Cell culture Growth medium DMEM 10 FBS 1 x NEAA 1 x Pen Strep Puromycin For generating stable pathway sensor cell lines Sigma cat p8833 Firefly Luciferase Assay System o Luciferase Assay System Promega cat E1500 o Steady Glo Luciferase Assay System Promega cat E2510 o Bright Glo Luciferase Assay System Promega cat E2610 Dual Luciferase Assay System o Dual Luciferase Reporter Assay System Promega cat E1910 o Dual Glo Luciferase Assay System Promega cat E2920 96 well white opaque flat bottom microtiter plate Luminometer e FACS flow cytometer fluorescent microscope or fluorometer V Protocol A Before you begin Cell type selection The Cignal lentiviral particles are pseudotyped with the VSV G envelope protein This allows efficient transduction of lentiviral particles containing the transcription factor responsive reporter gene firefly luciferase or GFP into most mammalian cells When working with a cell type for the first time it is recommended to optimize the conditions for efficie
32. urpose of producing useful or valuable proteins in the eggs of such transgenic birds the delivery of gene therapies and for commercial production of therapeutic diagnostic or other commercial products not intended for research use where such products do not consist of or incorporate a lentiviral vector Information about licenses for commercial uses excluded under this license is available from Oxford BioMedica UK Ltd Medawar Center Oxford Science Park Oxford OX4 4GA UK enquiries oxfordbiomedica co uk or BioMedica Inc 11622 El Camino Real 100 San Diego CA 92130 2049 USA LentiVector is a registered US and European Community trademark of Oxford BioMedica plc Fluorescent Protein Limited License Statement This product is for internal non commercial research use only No rights are conveyed to modify or clone the gene encoding fluorescent protein contained in this product The right to use this product specifically excludes the right to validate or screen compounds For information on commercial licensing contact Evrogen Licensing Department email license evrogen com Dual Glo Dual Luciferase Steady Glo Bright Glo and Monster Green are trademarks of Promega Corporation Opti MEM is a registered trademark of Life Technologies 2012 QIAGEN all rights reserved U o s MM www qiagen com Australia Orders 1 800 243 800 Fax 03 9840 9888 Technical 1 800 243 066 Austria Orders 0800 28 10 10 Fax 0800
33. w can make sure that my cell type of interest can be transduced with Cignal Lentiviruses A The Cignal Lentiviruses are pseudotyped with VSV G Protein which is pantropic and allows the lenitivirus to interact with its target cell in a receptor independent manner This receptor independent entry into the target cell likely involves endocytosis Thereby in theory the lentivirus can transduce virtually any mammalian cell type Also lentivirus does not require a mitotic event for integration into the host cell genome However it is recommended to consult the literature or utilize our Cignal Lenti positive control cat CLS PCG to determine if your target cells of interest can be transduced with Cignal Lentiviruses Q Can Cignal Lentivirus particles be further propagated in the lab A No Cignal Lentivectors are engineered for maximum biosafety and are therefore replication incompetent Genes for replication are not included in the packaged viral genome and the lentiviral vector contains a self inactivating 3 LTR Q Does the Cignal lentivirus produce any toxic viral genes A The Cignal Lentiviruses do not carry or express any viral genes and therefore have no associated toxicity issues Q How labile is the Cignal lentivirus A The lentivirus is sensitive to temperature 65 C or higher hypo osmolarity 1096 bleach 7096 ethanol and detergents Triton X 100 etc Q How one can decontaminate lentiviral contaminated surfaces A
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