User Manual - Biomol GmbH
Contents
1. 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 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 Lenti 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 Technical Support 888 503 3187 US 301 682 9200 122. Version 1 3 Reporter gene cppt hPGK TATA box RRE SIN 3 LTR pCignal Lenti minP Reporter gene Psi RSV 5 LTR AmpR Reporter gene cppt hPGK CMV SIN 3 LTR pCignal Lenti CMV Reporter gene RRE Psi RSV 5 LTR 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 8 for the pCignal Lenti TRE Reporter gene vectors AmpR Technical Support support SABiosciences com www SABiosciences com 13 Cigna I Lenti Reporters 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 Transduction Reagent SABiosciences cat SA 02 Cignal Lenti Negative Control o For firefly luciferase reporter studies SABiosciences cat CLS NCL o For GFP reporter studies SABiosciences cat CLS NCG Cignal Lenti Positive Control o For firefly luciferase reporter studies SABiosciences cat CLS PCL o For GFP reporter stud
3. 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 It 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 The Cignal Lenti Reporters work very well for transient transduction experiments In such transient pathway activation studies reporter gene expression is typically 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 30 of cells even when transduced at high MOl s For these difficult to transduce cells it is important to select the cells stably expressing the reporter gene by puromycin se
4. reproducible transduction results it is important to determine the cell density with 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 CO2 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 CO2 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 type for the first time a SureENTRY Transduction Reagent control only well should b
5. 10 reporter and control tubes total negative and positive controls 1 tube each Cignal Finder Lenti Toxicity Ready to transduce transcription gt 0 8 x10 TU ml 10 Pathway Reporter Array factor responsive lentiviral firefly 250 ul of each CLA 004L luciferase pathway reporters 10 reporter and control tubes total negative and positive controls 1 tube each Technical Support support SABiosciences com www SABiosciences com 25 Notes Notes Technical Support support SABiosciences com www SABiosciences com 26 Version 1 3 Technical Support support SABiosciences com www SABiosciences com 27 Cignal Lenti Reporters Part 1037A Version 1 3 3 20 2009 A SABiosciences Focus on Your Pathway BIOMOL GmbH Waidmannstr 35 O m 22769 Hamburg b m info biomol de 10 o www biomol de Phone 49 40 8532600 or 0800 2466651 D Fax 49 40 85326022 or 0800 2466652 D 888 503 3187 301 682 9200 www SABiosciences com support SABiosciences com
6. S saBiosciences writes CY biomol info biomol de www biomol de Phone 49 40 8532600 or 0800 2466651 D Fax 49 40 85326022 or 0800 2466652 D User Manual CEE Cignal Lenti Reporters Lentiviral Cell Signaling Pathway Reporters See Purchaser Notification for limited use license and warranty information pages 2 and 3 Part 1037A Version 1 3 3 20 2009 SABiosciences Corporation 6951 Executive Way Frederick MD 21703 USA Cignal Lenti Reporters Lentiviral Cell Signaling Pathway Reporters BIOMOL GmbH Waidmannstr 35 22769 Hamburg A biomol info biomol de U S e r M a n u al Prone A A0 U53200 or 0800 2466651 D Fax 49 40 85326022 or 0800 2466652 D For Catalog Numbers CLS L CLS G and CLA L Ordering and Technical Service Contact Information Tel 1 888 503 3187 US 301 682 9200 outside US Fax 1 888 465 9859 US 301 682 7300 outside US On line Order www SABiosciences com E MAIL order SABiosciences com to place an order support SABiosciences com for technical support You may place orders by fax e mail or from our website Each order should include the following information Your contact information name phone email address Product name catalog number and quantity Purchase order number or credit card information Visa or MasterCard Shipping address Billing address For more information visit us at www SABiosciences com LIMITED PRODUCT WARRANTY This wa
7. a polyadenylation signal for efficient transcription termination f1 ori f1 origin of replication Allows episomal replication of plasmid in eukaryotic cells 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 Technical Support support SABiosciences com www SABiosciences com 9 Cignal Lenti Reporters 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 and centrifuged the media 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 may vary for different lots and are provided on the certificate of analysis included in each shipment D Biosafety Features of Cignal Lentiviral Part
8. 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 like HT1080 HEK293 and HepG2 etc 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 Technical Support support SABiosciences com www SABiosciences com 15 Cignal Lenti Reporters 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 like this titration of SureENTRY Transduction Reagent using 2 4 6 8 ug ml will
9. arget of a specific signaling pathway Technical Support 888 503 3187 US 301 682 9200 8 Version 1 3 TATA box TRE Reporter gene cppt hPGK RRE SIN 3 LTR pCignal Lenti TRE Reporter gene Psi RSV 5 LTR AmpR Figure 2 Schematic of lentiviral vector used to generate Cignal Lenti Reporters 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
10. ay Regulatory Element Transcription Factor luciferase Cat No TRE Cat No C EBP C EBP binding C EBP CLS 001L element cAMP PKA cAMP regulatory CREB CLS 002L CLS 002G element CRE Cell Cycle E2F binding element E2F DP1 CLS 003L DNA Damage p53 response element p53 CLS 004L Hypoxia HIF response element Hypoxia inducible CLS 007L CLS 007G factor 1 HIF 1 Type Interferon Interferon stimulated STAT1 STAT2 CLS 008L response element ISRE Interferon Gamma Interferon gamma STAT1 STAT1 CLS 009L activation sequence GAS MAPK ERK Serum response Elk 1 SRF CLS 010L CLS 010G element SRE MAPK JNK AP1 binding element AP 1 CLS 011L CLS 011G c Myc E box binding element Myc Max CLS 012L NFkB NFB binding element NFkB CLS 013L CLS 013G Notch RBP J binding RBP Jic CLS 014L CLS 014G element PKC Ca NFAT response NFAT CLS 015L element Retinoic Acid Retinoic acid Retinoic Acid Receptor CLS 016L Receptor response element RAR RARE TGFB SMAD response SMAD2 SMAD3 SMAD4 CLS 017L element Wnt TCF LEF response TCF LEF CLS 018L CLS 018G element Technical Support 888 503 3187 US 301 682 9200 24 Version 1 3 Cignal Lenti Reporter Controls Control Construct Components Concentration Catalog Number And Volume Ready to transduce Cignal Lenti Negative lentivirus expressing non gt 0 8 x10 TU mI CLS NCL Control LUC inducible firefly luciferase 250 ul 1 tube Ready to transduce Cignal Lenti Positive lentiviru
11. e 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 SABiosciences Corporation 6951 Executive Way Suite 100 Frederick MD 21703 USA Technical Support support SABiosciences com www SABiosciences com 3 CONTENTS l Introduction 5 I Product Contents and Descriptions 8 A Contents 8 B Description 8 C Production and Titration of Cignal Lentiviral Particles 10 D Biosafety Features of Cignal Lentiviral Particles 10 E Safety Guidelines 10 Ill Cignal Lenti Reporter Controls 12 IV Additional Materials Required 14 V Protocol 15 A Before You Begin 15 B Brief Protocol 17 C Detailed Protocol 18 VI Frequently Asked Questions 21 Appendix Troubleshooting and Cignal Lenti Reporter Products 23 Technical Support support SABiosciences com www SABiosciences com 4 Version 1 3 I 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 SABiosciences Cignal Lenti Reporte
12. e used to determine cell sensitivity Technical Support 888 503 3187 US 301 682 9200 18 Version 1 3 Important When transducing Cignal lentiviral 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 wavelen
13. ed 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 reporter GFP enables you to monitor the dynamics of pathway activation on living cells with single cell resolution The Cignal Lenti reporter GFP 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 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 Technical Support support SABiosciences com www SABiosciences com 5 Cignal Lenti Reporters Benefits of Cignal Lenti Reporters Ready to tran
14. evelopment 8 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 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 9 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 Technical Support 888 503 3187 US 301 682 9200 20 Version 1 3 Q How many cells can transduce with the amount of Cignal Lenti Reporter provided by SABiosciences A The amount of cells that can be transduced depends upon your chosen target cells and how eas
15. g use and handling of the lentiviral system Technical Support 888 503 3187 US 301 682 9200 10 Version 1 3 While working with Cignal Lenti Reporters we also recommend following standard safety practices e Wear double gloves and lab coat at all times e Perform work in a Class II 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 Technical Support support SABiosciences com www SABiosciences com 11 pM Cignal Lenti Reporters lll Cignal Lenti Reporter Controls Control Construct Components Concentration Catalog Number And Volume Ready to transduce Cignal Lenti Negative lentivirus expressing non gt 0 8 x10 TU mI CLS NCL Control LUC inducible firefly luciferase 250 ul per tube Ready to transduce Cignal Lenti Positive lentivirus constitutively gt 0 8 x10 TU mI CLS PCL Control LUC expressing firefly luciferase 250 ul per tube Ready to transduce Cignal Lenti Renilla lentivirus constitutively gt 0 8 x10 TU ml CLS RCL Control Rluc expressing Renilla 250 ul per tube luciferase Ready to transduce Cignal Negative Control lentivirus expressing non gt 0 8 x10 TU mI CLS NCG GFP inducible GFP 250 ul per tube Ready to transduce Cignal Positive Control lentivirus constitutively gt 0 8 x10 TU mI CLS PCG GFP expressing GFP 250 ul per tube
16. gth 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 the effect of sIRNA ShRNA on a specific reporter or signaling pathway we recommend doing transient transfection of siIRNA 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 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 Technical Support support SABiosciences com www SABiosciences com 19 py Cignal Lenti Reporters 7 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 about 6 or 24 hours before the assay d
17. hRNA plasmids or expression vectors Day 4 or 5 Depending upon experimental design treat with test proteins peptides or compounds Day 5 or 6 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 Technical Support support SABiosciences com www SABiosciences com 17 pM Cignal Lenti Reporters C Detailed Protocol The following protocol is designed to transduce HEK293 cells using Cignal Lenti Reporters in a 96 well plate format The Cignal Lenti Reporters 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 x10 cells ml in growth media To ensure
18. icles The Cignal Lentiviral particles have numerous biosafety features which include e A deletion in the promoter enhancer region of the U3 portion 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 edition http www cdc gov od ohs biosfty bmbl4 bmbl4toc htm You should also consult the health and safety guidelines and officers at your institution regardin
19. ies SABiosciences cat CLS PCG Cignal Lenti Renilla Control For dual luciferase reporter assay format SABiosciences cat CLS RCL Cell culture Growth medium DMEM 10 FBS 1x NEAA 1x 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 FACS flow cytometer fluorescent microscope or fluorometer Technical Support 888 503 3187 US 301 682 9200 14 Version 1 3 V Protocol A Before you begin 1 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 efficient transduction 2 Optimization of conditions for efficient transduction The sensitivity of the Cignal Lenti Reporter Assay depends on the transduction efficiency The transduction efficiency in
20. ily they are transduced Primary or other difficult cells may require higher MOIl s 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 How can I 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 o
21. lection 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 Perform all transduction in triplicate to minimize variability among treatment groups 2 Include positive and negative controls in each experiment to obtain reliable results 3 Take care to always seed the same number of cells in each well in order to maximize the reproducibility of your experiment Serum induces various signaling pathways leading to cross talk and high background Therefore use reduced amounts of serum 0 5 in the assay medium during the experimental treatment to minimize these serum effects a Technical Support 888 503 3187 US 301 682 9200 16 Version 1 3 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 test sIRNA s
22. nd 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 suggest 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 Technical Support support SABiosciences com www SABiosciences com 23 Cignal Lenti Reporters Cignal Lenti Reporters Transcriptional Dual GFP Pathw
23. r 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 10 bleach 70 ethanol and detergents Triton X 100 etc Q How one can decontaminate lentiviral contaminated surfaces A Please follow CDC guidelines We typically use 10 bleach to inactivate the virus Q What does transduction unit TU mean Technical Support support SABiosciences com www SABiosciences com 21 py Cignal Lenti Reporters 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 Technical Support 888 503 3187 US 301 682 9200 22 Version 1 3 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 a
24. rranty limits our liability to replace this product in the event the product fails to perform due to any manufacturing defect SABiosciences Corporation makes no other warranties of any kind expressed or implied including without limitation warranties of merchantability or fitness for a particular purpose SABiosciences Corporation shall not be liable for any direct indirect consequential or incidental damages arising out of the use the results of use or the inability to use this product NOTICE TO PURCHASER This product is intended for research purposes only and is not intended for drug or diagnostic purposes or for human use The purchase of Cignal Lenti Reporters includes a limited nonexclusive license to use the components for research use only This license does not grant rights to use the components for reproduction of any components to modify any components for resale or to manufacture commercial products without written approval of SABiosciences Corporation No other license expressed implied or by estoppel is granted NOTICE TO PURCHASER II The Dual Luciferase Reporter Assay is a trademark of Promega Corporation NOTICE TO PURCHASER III 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
25. rs 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 of the Cignal Lenti reporters is available with either luciferase or GFP as a reporter gene The Cignal Lenti reporters 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 reporter luc encodes for the mammalian codon 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 measur
26. s constitutively gt 0 8 x10 TU mI CLS PCL Control LUC expressing firefly luciferase 250 ul per tube Ready to transduce Cignal Lenti Renilla lentivirus constitutively gt 0 8 x10 TU ml CLS RCL Control Rluc expressing Renilla 250 ul per tube luciferase Ready to transduce Cignal Negative Control lentivirus expressing non gt 0 8 x10 TU mI CLS NCG GFP inducible GFP 250 ul per tube Ready to transduce Cignal Positive Control lentivirus constitutively gt 0 8 x10 TU mI CLS PCG GFP expressing GFP 250 ul per tube Cignal Finder Lenti 10 Pathway Reporter Arrays Product Name Components Concentration and Catalog Number Volume Cignal Finder Lenti Cancer Ready to transduce transcription gt 0 8 x10 TU ml 10 Pathway Reporter Array factor responsive lentiviral firefly 250 ul of each CLA 001L luciferase pathway reporters 10 reporter and control tubes total negative and positive controls 1 tube each Cignal Finder Lenti Immune Ready to transduce transcription gt 0 8 x10 TU ml Response 10 Pathway factor responsive lentiviral firefly 250 ul of each CLA 002L luciferase pathway reporters 10 Reporter Array tubes total negative and reporter and control positive controls 1 tube each Cignal Finder Lenti Ready to transduce transcription 2 0 8 x10 TU ml Development 10 Pathway factor responsive lentiviral firefly 250 ul of each CLA 003L Reporter Array luciferase pathway reporters
27. sciences com www SABiosciences com 7 Cignal Lenti Reporters ll 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 _ anscription factor gt 0 8 x10 TU ml 250 pl responsive lentiviral reporter Ready to transduce Lenti Reporter 8 tubes nscription 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 12 B Description Cignal Lenti_ Reporter The Cignal Lenti Reporters are delivered as ready to transduce lentiviral particles expressing an inducible 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 The Cignal Lenti Reporters monitor both increases and decreases in the activity of a key transcription factor which is a downstream t
28. sduce Lentiviral vectors arrive as transduction ready lentiviral particles eliminating any need to construct and amplify lentivirus in your laboratory 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 lines 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 Technical Support 888 503 3187 US 301 682 9200 6 Version 1 3 Transient Establish Pathway Transduction Studies Sensor Cell Lines J Plate Cells Plate Cells l Cignal Lenti Reporter live lentivirus particles ba x m Transduction Y Transduction w Cignal Lenti l Xy 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 Reporters are ready for transduction right out of the box There is no need to generate or propagate lentivirus in your laboratory These vectors are useful for transient transduction studies in difficult to transfect cells or for pathway sensor cell line generation Technical Support support SABio
29. 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 Version 1 3 NOTICE TO PURCHASER III continued 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 purpose 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 incorporat
30. 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 SABiosciences 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 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
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