User Manual - System Biosciences
Contents
1. PL Nolan GP Efficient transduction of nondividing cells by optimized feline immunodeficiency virus vectors Mol Ther 2000 Jan 1 1 31 8 Poeschla EM Wong Staal F Looney DJ Efficient transduction of nondividing human cells by feline immunodeficiency virus lentiviral vectors Nat Med 1998 Mar 4 3 354 7 Poeschla E M Looney D J and Wong Staal F 2003 Lentiviral nucleic acids and uses thereof US Patent NO 6 555 107 B2 Dull T Zufferey R Kelly M Mandel R J Nguyen M Trono D 1998 J Virol 72 8463 8471 Miyoshi H Blomer U Takashi M Gage F N Verma I M 1998 J Virol 72 8150 8157 Zufferey R Donello J E Trono D Hope T J 1999 J Virol 73 2886 2892 Ramezani A Hawley T S Hawley R G 2000 Mol Ther 2 458 469 Leung T H Hoffmann A Baltimore D 2004 Cell v 118 453 464 888 266 5066 Toll Free 650 968 2200 outside US Page 13 System Biosciences SBI User Manual V Appendix A pGF1 CMV Features RSV 5 LTR 1 415 Hybrid RSV promoter R US long terminal repeat PT RR for viral packaging and transcription 567 919 Packagingsigna sid 4066 1309 Rev response element binds gag and involved in packaging of viral transcripts Central polypurine tract includes DNA Flap cPPT 1798 1899 region involved in nuclear translocation and integration of transduced viral genome Human cytomegalovirus CMV constitutive OMV promoter 19433221A promoter for transcriptio2. Tat independent production of viral RNA reducing the number of genes from HIV 1 that are used in this system Number of HIV 1 viral genes necessary for packaging replication and transduction is reduced to three gag rev and pol and these genes are expressed from different plasmids lacking packaging signals and significant homology to pGF1 expression vector VSV G expression vector or each other to prevent generation of recombinant replication competent virus None of the HIV 1 genes gag pol rev will be present in the packaged viral genome as they are expressed from packaging plasmids lacking packaging signal therefore the lentiviral particles generated are replication incompetent Pseudoviral particles will carry only the expression construct of your target gene To avoid any possible contamination and maintain a clean laboratory environment we also recommend following these standard safety practices Wear double gloves face protection and lab coat at all times Perform work in a limited access area in a Biological Safety Cabinet Class Il and post biohazard warning signs Minimize splashes or aerosols with careful pipetting Take precautions with needles blades etc Decontaminate work surfaces at least once a day and after any spill of viable material Decontaminate all cultures stocks and other biological wastes before disposal using approved decontamination methods such as autoclaving Before decontamination the biolo
3. Wong Staal F 2000 Development of lentiviral vectors for gene theraphy for human diseases Blood 95 2499 2504 Burns J C Friedmann T Driever W Burrascano M and Yee J K 1993 Vesicular stomatitis virus G glycoprotein pseudotyped retroviral vectors concentration to a very high titer and efficient gene transfer into mammalian and non mammalian cells Proc Natl Acad Sci USA 90 8033 8034 Cann A J ed 2000 RNA Viruses A Practical Approach Oxford Univ Press Dull T Zufferey R Kelly M Mandel R J Nguyen M Trono D and Naldini L 1998 A third generation lentivirus vector with a conditional packaging system J Virol 72 8463 8471 Gould D J and Favorov P 2003 Vectors for the treatment of autoimmune diseases Gene Therapy 10 912 927 Morgan R A Cornetta K and Anderson W F 1990 Application of the polymerase chain reaction in retroviral mediated gene transfer and the analysis of gene marked human TIL cells Hum Gene Ther 1 135 149 Pfeifer A Kessler T Yang M Baranov E Kootstra N Cheresh D A Hoffman R M and Verma I M 2001 Transduction of liver cells by lentiviral vectors Analysis in living animals by fluorescence imaging Mol Ther 3 319 322 Qin X F An D S Chen I S and Baltimore D 2003 Inhibiting HIV 1 infection in human T cells by lentiviral mediated delivery of small interfering RNA against CCR5 Proc Natl Acad Sci USA 100 183 188 Quinn T P a
4. have been successfully transduced with lentivectors please see the Reference Section The pseudoviral particles can infect or transduce target cells and express reporter gene but cannot replicate within target cells because the viral structural genes are absent Following transduction into the target cells the GF cassette is reverse transcribed and integrated into the genome of the target cell After integration the GF cassette is under the transcriptional control of the transcriptional regulatory response elements and dscGFP luciferase expression can be activated under the appropriate conditions Page 4 ver 3 140110 www systembio com pGreenFire Plasmid and Virus Response Reporter Constructs Cat SR20070 SR20071 Freeze cells for future use Monitor trans activation using GFP Fluorescence microscopy Flow cytometry Microplate reader or Firefly Luciferase eeoeccc 2 520 Luminometer 00000000 00000000 000006000 ooeooo000 0000000 000000 0000000 0000000 0000000 00000600 0000000 000000 Summary of Workflow for pGreenFire1 Lentiviral Reporter Vectors Packaged virus is used to transduce infect cells of interest and introduce the reporter construct into the cell as an integrated provirus Transduced cells can be split and 1 assayed for reporter activation 2 frozen as a stock of cells for future use or 3 cloned to obtain monoclonal populations with more uniform background and activation levels Oct4 Sox2 Respo
5. htm Also you should consult the health and safety guidelines and officers at your institution regarding use and handling of the lentiviral system In addition although the system itself has been designed to minimize possible risk specific recombinant lentivector constructs may be potentially hazardous depending on the nature of introduced insert such as oncogenes toxins Transcriptional Response Element to tumor suppressor genes etc For these reasons it is critical to exercise due caution while working with recombinant lentiviruses To ensure safe laboratory handling you should thoroughly understand the biology of the lentiviral vectors and the specific modifications and design features of the SBI system with which you are working Page 6 ver 3 140110 www systembio com pGreenFire Plasmid and Virus Response Reporter Constructs Cat SR20070 SR20071 SBI s pGF1 lentivectors together with the pPACKH1 packaging plasmids comprises a third generation HIV 1 based cloning vector system These lentivectors are based on the vectors developed for gene therapy applications by Dr J G Sodroski US patent 45 665 577 and 5 981 276 This system is designed to maximize its biosafety features including Deletion in the enhancer of U3 region of 3 LTR ensures self inactivation of lentiviral construct after transduction and integration into genomic DNA of the target cells RSV promoter upstream of 5 LTR in pGF1 expression vector allows efficient
6. lines such as HEK293T use 3 FBS in the medium Add Polybrene to a final concentration of 5 8 ug ml Note Polybrene is a polycation that neutralizes charge interactions to increase binding between the pseudoviral capsid and the cellular membrane The optimal concentration of Polybrene depends on cell type and may need to be empirically determined usually in the range of 2 10 ug ml Excessive exposure to Polybrene gt 12 hr can be toxic to some cells 3 Remove test tube with packaged pGF 1 reporter vector from 70 C and place directly in ice Thaw the viral particles for 5 10 min Dilute an appropriate amount depending on the optimal MOI of viral particles in 0 1 ml of complete D MEM medium with Polybrene from step 2 Use several dilutions of packaged pGF reporter vector if necessary Note We recommend diluting the viral particles in a smaller volume of medium if possible The higher the titer of virus in solution the higher is the transduction efficiency Mix the virus with the medium gently by rotation or inversion Do not vortex 4 Remove the culture medium from cells Infect HT1080 and target cells by adding the viral stock dilutions to the wells For one well mock well control add 0 1 ml of D MEM medium with Polybrene from Step 2 Incubate cells at 37 C with 5 CO overnight Day 3 5 Remove the culture medium and replace with 0 5 ml of complete D MEM medium without Polybrene but with serum and antibiotics Inc
7. work in stem cells choose the MSCV option if you are working with stem cells The negative control construct with mCMV promoter TRO10VA 1 are available separately The Packaged Lentiviral Reporter Vectors are shipped on dry ice and should be immediately stored at 70 C upon receipt Avoid thawing and refreezing of pseudoviral particles Properly stored pseudoviral particles are stable for 6 months from the date received F Additional Required Materials e Dulbecco s Modified Eagle s Medium D MEM high glucose with sodium pyruvate and glutamine Invitrogen Cat 11995073 Fetal Bovine Serum Invitrogen Cat 16000036 Penicillin Streptomycin Invitrogen Cat 15070063 Trypsin EDTA Sigma Cat T3924 Polybrene hexadimethrine bromide Sigma Cat H9268 Millex HV 0 45 um PVDF filters Millipore Cat SLHVR25LS Tissue Culture Plates and Related Tissue Culture Supplies 293TN Human Kidney Producer Cell Line SBI Cat LV900A 1 Lipofectamine and Plus Reagent Invitrogen Third generation HIV based packaging plasmids such as SBI pPACK H1 LV500A 1 G Safety Guidelines Work with HIV based lentiviral vectors falls within NIH Biosafety Level 2 criteria For a detailed description of laboratory biosafety level criteria consult the following pages on the Centers for Disease Control Office of Health and Safety Web site http www cdc gov od ohs biosfty bmbl4 bmbl4s3 htm http www cdc gov od ohs biosfty bmbl4 bmbl4toc
8. SBI System Biosciences Pluripotency Reporters Cat SR2007x Series User Manual Store at 20 C A limited use label license covers this product By use of this product you accept the terms and conditions outlined in the Licensing and Warranty Statement contained in this user manual pGreenFire Plasmid and Virus Response Reporter Constructs Cat SR20070 SR20071 Contents l Introduction and Background A Overview sss lulu 2 B Lentiviral Expression System 2 C Production of Pseudotyped Viral Particles 4 D Delivery of Packaged Vector Construct into Target Cells 4 E List of Components and Available Reporters 6 F Additional Required Materials 6 G Safety Guidelines sss 6 ll Protocol A Procedure Outline and General Comments 8 B Transduction of Packaged pGF1 Reporter Vector 9 lll Troubleshooting 11 IV References ss 12 V Appendix A pGF1 mCMV Puro Features 13 B Properties of dscGFP Fluorescent Protein 14 C Related Products sss 14 D Technical Support sss 14 vi Licensing and Warranty Statement 16 This Product shall be used by the purchaser for internal research purposes only and distribution is strictly prohibited without written permission by System Biosciences 888 266 5066 Toll Free 650 968 2200 outside US Page 1 System Biosciences SBI User Manual I Introduction and B
9. ackground A Overview This manual provides information describing how to use the packaged VSV G pseudotyped lentiviral pGreenFire Transcriptional Reporter TR Constructs to generate stable cell lines with the reporter constructs integrated into the host genome Before using the reagents and material supplied with this product please read the entire user manual B Lentiviral Transcriptional Reporter System Eukaryotic gene expression is regulated by a wide variety of developmental and environmental stimuli First an extracellular signaling molecule binds to a specific receptor The signal is then transmitted through a series of molecular cascades which activate or deactivate specific transcription factors TFs that regulate gene expression The expression of any given gene is controlled by multiple transcription factors which in turn are modulated by multiple signal transduction pathways Many of these signal transduction pathways converge at transcription factors that bind to specific transcriptional response elements TREs or regions found in the promoters of various genes and modulate the transcription of these genes The activation of a signal transduction pathway e g by growth factors drugs etc can therefore be monitored by the expression level of the reporter gene controlled by a promoter containing these response sequences The commonly used plasmid based transcriptional reporter vectors containing different reporter genes can be d
10. cGFP protein is the presence of an additional destabilizing ds peptide on the C end of the protein which shortens the half life time of the mature protein without additional transcription to 1 hour The copGFP protein is a non toxic non aggregating protein with fast protein maturation high stability at a wide range of pH pH 4 12 and does not require any additional cofactors or substrates The copGFP protein has very bright fluorescence that exceeds at least 1 3 times the brightness of EGFP the widely used Aequorea victoria GFP mutant The copGFP protein emits green fluorescence with the following characteristics emission wavelength max 502 nm excitation wavelength max 482 nm quantum yield 0 6 extinction coefficient 70 000 M cm Due to its exceptional properties copGFP is an excellent fluorescent marker which can be used instead of EGFP for monitoring delivery of lentiviral constructs into cells C Related Products e PathNet Lentiviral TRE Cloning and Delivery Vectors gt pTRF1 mCMV dscGFP Vector Cat TR100PA 1 gt pTRF3 mCMV Luc Vector Cat TR200PC 1 gt pTRH1 mCMV dscGFP Vector Cat TR500PA 1 gt pTRH5 mCMV Luc Vector Cat TR600PE 1 These FIV and HIV based Lentiviral Transcriptional Reporter cloning vectors allow you to clone transcriptional response elements TRE under the mCMV promoter or promoter of your choice and efficiently transduce these constructs in a wide range of cells
11. e PathNet Lentiviral Transcriptional Reporter Constructs in Plasmid form Many visit our website at www systembio com for a current list of available TR plasmid constructs These FIV and HIV based plasmid reporter constructs when packaged in pseudoviral particles using the pPACKF 1 or pPACKH 1 Lentivector Packaging Kit allow you to transduce and analyze a wide variety of TF specific constructs in a wide range of cells Based on the highly efficient lentiviral system the PathNet Transcriptional Reporter Vectors provide a convenient and cost effective system to deliver and stably integrate sequences of your choice into the host genome e pPACK Lentivector Packaging Kits gt FIV Based pPACKF1 Packaging Kit Cat LV100A 1 gt HIV Based pPACKH1 Packaging Kit Cat LV500A 1 Unique lentiviral vectors that produce all the necessary lentiviral proteins and the VSV G envelope glycoprotein from vesicular stomatitis virus required to package pGF1 lentiviral constructs into pseudoviral particles 293TN cells SBI Cat LV900A 1 or ATCC Cat CRL 11268 transiently transfected with the Lentiviral Packaging Plasmid Mix and one of the pGF1 reporter vectors produce packaged pseudoviral particles containing a pGF1 TRE mCMV construct e Lentivector UltraRapid Titer PCR Kit Cat LV960A 1 for human cells LV961A 1 for mouse cells Measure copy number MOI of integrated lentiviral constructs in genomic DNA of target cells after transd
12. elivered by transient transfection to the nucleus of target cells to monitor the activation of signal transduction pathways converging at a specific response element Lentiviral expression vectors are the most effective vehicles for delivering genetic material to almost any mammalian cell including non dividing cells and to model organisms As with standard plasmid vectors it is possible to introduce lentiviral Transcriptional Reporter TR constructs in plasmid form into the cells with low to medium efficiency using conventional transfection protocols However by packaging the lentiviral TR vector construct in pseudoviral particles you can obtain highly efficient transduction and heritable expression of transcriptional reporter constructs even with the most difficult to transfect cells like primary stem and differentiated cells In comparison to retroviral delivery systems lentivectors enter the cell nucleus without requiring cell replication Page 2 ver 3 140110 www systembio com pGreenFire Plasmid and Virus Response Reporter Constructs Cat SR20070 SR20071 Advantages of lentivector technology include Fig Ready to use pre packaged constructs with a wide range of Transcriptional Response Elements TREs or regions for multiple transcriptional factors Lentiviral reporter constructs can efficiently transduce nearly all cell types even those that are difficult to transfect such as primary or non dividing mammalian cells Our le
13. enous viral sequences to form a self replicating HIV virus In addition the pGF1 cloning vectors developed at SBI are self inactivating as a result of a deletion in U3 region of 3 LTR Upon integration into the genome the 5 LTR promoter is inactivated which prevents formation of replication competent viral particles The pGF1 vectors express dscGFP destabilized reporter gene and Firefly luciferase_under the control of a minimal CMV mCMV promoter linked to transcriptional regulatory elements TRE or sequences such as Oct4 conserved region 4 The WPRE element enhances the expression level of the reporter gene Constitutively expressed Puro Neo resistant gene allows efficieny selection of stable transfectants Figure 1 shows a map of the pGF1 mCMV vector that expresses green fluorescent protein and Firefly luciferase from the mCMV promoter and can be used as a negative control In order to facilitate the use of the lentivector based GF system SBI offers a wide range of transcriptional reporter constructs packaged in VSV G pseudoviral particles Nde RSV Oct4 CR4 or Sox2 SSR2 S LTR y Response Elements Amp Pluripotency Response Reporters pUC ori SV40 ORI 5V40 Poly A 3 ALTI V yppg Luciferase Optional constitutive EF1 Puro or Neo Marker for easy Cell Line Construction 1 Map of the HIV based pGreenFire Pluripotency Response Reporter Vector Essential components of the pGF1 vector system are Response Ele
14. gical materials should be placed in a sealed durable leak proof container for transport from the laboratory 888 266 5066 Toll Free 650 968 2200 outside US Page 7 System Biosciences SBI User Manual Protocol A Procedure Outline and General Comments Some key terms used in the protocol MOI multiplicity of infection the average copy number of lentiviral expression constructs per genome of target cell in the infected cell population Pseudoviral titer ifu ml The relative titer concentration infection units ml of lentiviral constructs measured by amplification of the lentivector specific WPRE region from genomic DNA of HT1080 infected cells As a calibration standard we use DNA from cells infected with a GFP reporter construct at different multiplicity of infection MOI based on FACS analysis of the percentage of GFP positive cells The Pseudoviral Titer is always specific to a particular cell line To ensure optimal results follow these general guidelines during your experiments Page 8 pGF1 CMV Reporter Construct This plasmid should be used to estimate transduction efficiency of the lentiviral expression construct into target cells select the cell type with highest infection efficiency and to optimize the transduction protocol Moreover the presence of dscGFP positive cells indicates that the lentiviral construct can be efficiently expressed in your target cells from the CMV promoter The construct can also used f
15. ing storage Ensure storage of the Packaged Reporter Vector at 70 C Each freeze thaw cycle causes reduction of the titer by 20 3096 Use a fresh stock for transduction Do not keep the stock longer than 6 12 months Volume of infecting supernatant is too high Keep the volume as low as possible to achieve maximal adsorption of viral particles to the cells 2 Transduction affects target cell viability Packaged Reporter Vector affects target cell growth Use a shorter transduction time to minimize the toxic effect to the target cells Try replacing with a similar target cell type Polybrene is toxic for target cells Optimize the concentration and exposure time to Polybrene during the transduction step 3 No Expression of positive control pGF1 CMV reporter in target cells The CMV promoter is not functional in target cells It is a very rare case but the only way to solve this problem is to change the type of target cells 888 266 5066 Toll Free 650 968 2200 outside US Page 11 System Biosciences SBI User Manual IV References Okumura Nakanishi S Saito M Niwa H Ishikawa F Oct 3 4 and Sox2 regulate Oct 3 4 gene in embryonic stem cells J Biol Chem 2005 Feb 18 280 7 5307 17 Tomioka M Nishimoto M Miyagi S Katayanagi T Fukui N Niwa H Muramatsu M Okuda A Identification of Sox 2 regulatory region which is under the control of Oct 3 4 Sox 2 complex Nucleic Acids Res 2002 Jul 15 30 14 3202 13 Buchschacher G L and
16. ls shown in the top panel As a negative control cells transduced with the pGF1 mCMV control vector were also treated with TNF a Page 10 ver 3 140110 www systembio com pGreenFire Plasmid and Virus Response Reporter Constructs Cat SR20070 SR20071 Ill Troubleshooting A Why are my cells green following transfection for viral packaging In order to produce the viral genomic RNA that will be packaged into viral particles a strong promoter is included in the 5 LTR to drive its expression Due to the mechanism of lentiviral replication this promoter in the 5 LTR is not present in the genomic RNA or the resulting integrated provirus Therefore GFP is expressed from the viral 5 LTR following transfection however following transduction transcription should only occur from the reporter gene when activated B Inefficient Transduction of Packaged pGF1 Reporter Vector into Target Cells 1 Poor infection efficiency Target cells have too high or too low density Plate fewer or more cells in order to have about 50 confluency at infection stage Target cell line may be difficult to transduce Use a higher concentration less fold dilution of pseudoviral particles Optimize the transduction protocol and use as positive control cells H1299 cell line Wrong amount of Polybrene added during infection stage If Polybrene is toxic to the target cells optimize Polybrene concentration in the range of 1 5 ug ml Loss of pseudoviral titer dur
17. ments arrow mCMV promoter purple destabilized copGFP green the self cleaving T2A peptide yellow and the Firefly luciferase gene red and a selection resistance genes Puro or Neo Marker 888 266 5066 Toll Free 650 968 2200 outside US Page 3 System Biosciences SBI User Manual C Production of Pseudotyped Viral Particles Currently the most efficient technology for producing a high titer of infectious replication incompetent lentiviral particles is based on transient coordinated expression of a viral effector expression construct and plasmids carrying all the necessary packaging proteins delivered into packaging also called producer cells like HEK 293T by simultaneous transfection with lentiviral expression and packaging plasmids For more details on the packaging procedure see the pPACK H1 Lentivector Packaging Kit user manual available on SBI s website at www systembio com The pGF1 Response Reporter vectors can be packaged into VSV G pseudotyped viral particles by co transfection with the pPACK H1 Lentiviral Packaging Plasmid Mix into HEK 239T cells SBI 293TN Producer Cell Line Cat LV900A 1 Following transfection the media contains the pseudoviral particles and can be concentrated by PEGit precipitation or ultracentrifugation using the protocol described in the Lentivector Packaging Kit Cat LV500A 1 When expressed the hybrid RSV 5 LTR HIV based drives high level transcription of the lentiviral c
18. n of dscGFP and zeoR Copepod green fluorescent protein similar to regular EGFP but with brighter color as a DscGFP T2A FLuc 2329 4848 reporter for the transfected transduced cells a destabilizing ds peptide on the C end shortens the half life time of the mature protein to 1 hour WPRE 4859 5399 Posttranscriptional regulatory element which enhances the stability of the viral transcripts Required for viral reverse transcription self 3 ALTR AU3 5538 5771 inactivating 3 LTR with deletion in U3 region s prevents formation of replication competent viral particles after integration into genomic DNA SV40 Poly A 5843 5974 Transcription termination and polyadenylation SV40 Ori 5983 6129 Allows for episomal replication of plasmid in eukaryotic cells pUC Ori 6499 7172 C Allows for high copy replication in E coli AmpR 7317 8177 C Ampicillin resistant gene for selection of the plasmid in E coli The notation C refers to the complementary strand Page 14 ver 3 140110 www systembio com pGreenFire Plasmid and Virus Response Reporter Constructs Cat SR20070 SR20071 B Properties of the dscGFP Fluorescent Protein The pGF Vectors contain the full length copGFP gene with optimized human codons for high level of expression of the fluorescent protein from the CMV promoter in mammalian cells The copGFP marker is a novel natural green monomeric GFP like protein from copepod Pontellina sp A unique feature of the ds
19. nd Trevor K T 1997 Rapid quantitation of recombinant retrovirus produced by packaging cell clones Biotechniques 23 1038 1044 Sui G Soohoo C Affar E B Gay F Forrester W C and Shi Y 2002 A DNA vector based RNAi technology to suppress gene expression in mammalian cells Proc Natl Acad Sci U S A 99 5515 5520 Curran MA Nolan GP Nonprimate lentiviral vectors Curr Top Microbiol Immunol 2002 261 75 105 Curran MA Nolan GP Recombinant feline immunodeficiency virus vectors Preparation and use Methods Mol Med 2002 69 335 50 Loewen N Barraza R Whitwam T Saenz DT Kemler Poeschla EM FIV Vectors Methods Mol Biol 2003 229 251 71 Naldini L Lentiviruses as gene transfer agents for delivery to non dividing cells Curr Opin Biotechnol 1998 Oct 9 5 457 63 Sauter SL Gasmi M FIV vector systems Somat Cell Mol Genet 2001 Nov 26 1 6 99 129 Alisky JM Hughes SM Sauter SL Jolly D Dubensky TW Jr Staber PD Chiorini JA Davidson BL Transduction of murine cerebellar neurons with recombinant FIV and AAV5 vectors Neuroreport 2000 Aug 21 11 12 2669 73 Brooks Al Stein CS Hughes SM Heth J McCray PM Jr Sauter SL Johnston JC Cory Slechta DA Federoff HJ Davidson BL Functional correction of established central nervous system deficits in an animal model of lysosomal storage disease with feline immunodeficiency virus based vectors Proc Natl Acad Sci U S A 2002 Apr 30 99 9 6216 21 Crystal RG Bad f
20. nd infected at 1 100 5 100 1 10 1 4 and 1 2 multiplicity of infection MOI and compared these results to FACS analysis of GFP positive cells The transduction efficiency of the pGF1 Packaged Reporter Construct and your lentiviral expression construct varies significantly for different cells and experimental conditions In order to optimize transduction conditions we recommend that you use HT1080 or similar cells as a positive control in parallel with your target cells and use prepackaged pGF1 CMV TRO11VA 1 from SBI To determine the desired multiplicity of infection MOI appropriate for your target cells you should do several transductions with packaged pGF1 pseudoviral particles at different MOI s e g from 0 1 to 5 Results of these test transductions should be used to determine an optimal MOI that yields the optimal percentage of infected cells based on the percentage of cells expressing the GFP marker Note that some cell types such as primary tissue cultures of differentiated cells e g epithelial cells attached to a membrane etc may be resistant to infection regardless of the MOI Conventional cell cultures usually do not have infection resistant cells Expression of the pGF1 Reporter can be measured directly at about 48 72 hours after transduction At this time GF constructs are integrated into the genomic DNA resulting in stably transduced reporter cell lines Depending on the percentage of infected cells one or more copies of
21. nse Reporters Oct4 CCR4 Sox2 SRR2 pow Sample GFP data for both the Oct 4 CR4 and Sox2 SSR2 Response reporters in Human iPS Cells 888 266 5066 Toll Free 650 968 2200 outside US Page 5 System Biosciences SBI User Manual E List of Components The pGreenFire Response Reporter vectors are available as either plasmid DNA or pre packaged VSV G pseudotyped lentiviral particles Premade reporters for a variety of signaling pathways are available and come complete with positive pGF1 CMV and negative pGF 1 mCMV controls Ten micrograms of each plasmid DNA is provided IMPORTANT Please note that pGreenFire lenti reporter constructs must be transduced into target cells as a packaged virus in order for the constructs to function properly Transfection of the constructs into a target cell keeps the constitutive RSV promoter intact thus overriding the pathway specific TRE promoters and leading to nonspecific expression of the reporter genes Pre packaged pGF1 constructs are provided as frozen pseudoviral particles The total number of infection units ifu and concentration the titer were determined using HT1080 cells and may vary for different lots of each packaged reporter vector The exact ifu titer and volume for each packaged reporter construct are indicated on its corresponding Product Analysis Certificate For pre packaged virus the positive control construct with full CMV or MSCV promoters The CMV promoter does Not
22. ntiviral based reporter system is a novel approach to study transcriptional regulation and offers many advantages over current transcription reporter systems TR constructs will integrate into the genome and therefore be subject to chromatin regulation Leung T H et al 2004 Expression of the reporter gene indicates activation of a given transcriptional response element TRE by the cognate transcriptional factor in the natural chromosomal environment rather than in the episomal state in the nucleoplasm as is the case for conventional plasmid based TR vectors Tandem copies of integration can be avoided thus allowing for faithful promoter regulation Copy number of reporter constructs can be controlled by varying the multiplicity of infection MOI Construction of stable reporter cell lines is possible with TR lentivectors in just several days with a built in constitutively expressing Puro or Neo resistance gene Monitoring of signaling pathways by flow cytometry FACS is enabled by GFP reporters SBI s pGreenFire1 lentivectors are based on the traditional HIV Human immunodeficiency virus vector backbone To address biosafety issues SBI uses a third generation HIV lentiviral vector Dull T et al 1998 Miyoshi H et al 1998 Zufferey R et al 1999 Ramezani A et al 2000 In spite of improved biosafety features third generation HIV cloning vectors still pose a potential biohazard risk due to the possible recombination with endog
23. onstruct and produces a transcript that contains all the necessary functional elements e Psi RRE and cPPT for efficient packaging When this construct is expressed in HEK 293 cells that also express viral coat proteins e a packaging cell line the lentiviral transcripts are efficiently packaged into pseudoviral particles After isolation these pseudoviral particles containing the RNA version of the TR cassette can be efficiently transduced into any mammalian target cells Following transduction into the target cells this expression cassette is reverse transcribed and integrated into the genome of the target cell D Delivery of Packaged Vector Construct into Target Cells Pantropic VSV G pseudotyped viral particles containing the RNA copy of the pGF1 construct can be efficiently used to deliver and express reporter gene in a wide range of mammalian target cells Pseudotyped packaged pGF1 constructs can effectively transduce both dividing and non dividing established cell lines including primary and differentiated cells and numerous cell types neuronal dendritic endothelial retinal pancreatic hepatic aortic smooth muscle airway epithelia skin fibroblast macrophage cells etc Lentivectors have been successfully used for direct in vivo delivery and expression of transgenic constructs in hamster muscle mouse and primate brain rabbit and mouse airway epithelium and mouse liver For a more complete list of cells or tissues which
24. or calibration of FACS machine for maximum intensity of expression Please note this reporter construct is not suitable for stem cells due to the inactivation CMV promoter Use pGF1 MSCV promoter pGF1 mCMV Reporter Construct Negative control construct which can be used to transduce target cells under the conditions optimized for the positive control pGF1 CMV construct and determine background of GFP fluorescence of target cells with a non activated CMV promoter Infection with Polybrene Polybrene is a polycation that increase transduction efficiency by neutralizing charge interactions and increase binding between the pseudoviral capsid and the cellular membrane The optimal concentration of Polybrene depends on cell type and may need to be empirically determined usually in the range of 4 8 ug ml Excessive exposure to Polybrene gt 12 hr can be toxic to some cells Pseudoviral titer The titer of packaged pGF1 reporter constructs can be determined using a PCR based approach such as SBI s UltraRapid titer kit LV960A 1 Following transduction of pseudoviral particles into HT1080 cells and analysis of percentage of infected cells by amplification of pGF1 specific products from genomic DNA with gene specific primers the titer can be estimated by comparison to a known set of standards As a calibration control standard we used the genomic DNA control template isolated from HT1080 cells transduced with positive control pGF1 CMV construct a
25. or cats good for humans Modified feline immunodeficiency virus for gene therapy J Clin Invest 1999 Dec 104 11 1491 3 Curran MA Kaiser SM Achacoso PL Nolan GP Efficient transduction of nondividing cells by optimized feline immunodeficiency virus vectors Mol Ther 2000 Jan 1 1 31 8 Derksen TA Sauter SL Davidson BL Feline immunodeficiency virus vectors Gene transfer to mouse retina following intravitreal injection J Gene Med 2002 Sep Oct 4 5 463 9 Haskell RE Hughes SM Chiorini JA Alisky JM Davidson BL Viral mediated delivery of the late infantile neuronal ceroid lipofuscinosis gene TPP I to the mouse central nervous system Gene Ther 2003 Jan 10 1 34 42 Page 12 ver 3 140110 www systembio com pGreenFire Plasmid and Virus Response Reporter Constructs Cat SR20070 SR20071 Price MA Case SS Carbonaro DA Yu XJ Petersen D Sabo KM Curran MA Engel BC Margarian H Abkowitz JL Nolan GP Kohn DB Crooks GM Expression from second generation feline immunodeficiency virus vectors is impaired in human hematopoietic cells Mol Ther 2002 Nov 6 5 645 52 Stein CS Davidson BL Gene transfer to the brain using feline immunodeficiency virus based lentivirus vectors Methods Enzymol 2002 346 433 54 Browning MT Schmidt RD Lew KA Rizvi TA Primate and feline lentivirus vector RNA packaging and propagation by heterologous lentivirus virions J Virol 2001 Jun 75 11 5129 40 Curran MA Kaiser SM Achacoso
26. the GF construct may be integrated into the genomic DNA of each transduced cell These reporter cells can be cloned in order to obtain a uniform population of the GF cell line where the GF construct is integrated into a defined chromosomal location Some actively dividing cells e g 293 HT1080 HeLa etc may express the reporter construct in 80 90 of the cells after transduction at an MOI of 1 2 For these easy to transduce cells most biological assays can be performed at 48 72 hours after transduction However some primary cells may only express the pGF1 construct in 10 50 of cells even when transduced at high MOl s For these difficult to transduce cells it is probably desirable to select the cells stably expressing the control pGF1 CMV vector or your lentiviral expression construct by FACS for experimental assays or to obtain cloned populations of TR cell lines ver 3 140110 www systembio com pGreenFire Plasmid and Virus Response Reporter Constructs Cat SR20070 SR20071 e Due to the specificity of the pGF1 reporter GFP expression is only expected to occur in conditions where the particular signaling pathway is active For instance the pGF1 NF kB reporter is silent in infected cells until an activating stimulus is given such as TNF a then both dscGFP and firefly luciferase is co expressed e SBPs lentiviral GF constructs contain a deletion in the 3 LTR which leads to self inactivation of the lentiviral vector after integra
27. tion into genomic DNA Although more than one copy of a lentiviral construct may be integrated into the genome of a single cell the lentiviral construct cannot produce infectious viral particles However in spite of these safety features please remember that you are working with transducible pseudoviral particles Although the particles are replication incompetent they are infection competent so the lentiviral expression cassette which they carry will infect integrate and express in any mammalian cells Please follow the recommended guidelines for working with BSL 2 class viruses see Section I G for more details B Transduction of the Packaged pGF1 Reporter Vector The following protocol describes the general procedure for the transduction of the pGF1 Reporter Constructs packaged in pseudotyped viral particles into HT1080 cells This protocol assumes that you will use these guidelines in order to perform transduction of your target cells in parallel using HT1080 cells as a positive control and can be used as a starting point for the optimization for transduction of your particular cell type Day 1 1 Plate HT1080 cells and target cells in a 48 well plate at a density of 1x10 cells per well 24 hours prior to viral infection Add 0 5 ml of complete D MEM medium with serum and antibiotics and incubate cells at 37 C with 5 CO overnight Day 2 2 Prepare 10 ml of complete D MEM medium For extremely fast growing and metabolizing cell
28. ubate the cells at 37 C with 5 CO overnight Day 4 6 By day 4 the culture will be confluent Split it 1 3 to 1 5 depending on the type of cells and continue incubating for 48 hours in complete D MEM Day 6 7 The infected HT1080 cells target cells can be analyzed for expression of the pGF1 CMV and pGF1 reporter construct by fluorescent microscopy or by performing a luciferase assay Figure 2 demonstrates the activation of the NF KB specific reporter pGF1 NF KB TRO12A 1 following infection of 293T cells and activation with TNF a 888 266 5066 Toll Free 650 968 2200 outside US Page 9 System Biosciences SBI User Manual pGreenFirel NFkB Epifluorescence Microscopy 293TN cells were transduced with pGF1 NF B and treated with the indicated amount of TNFa for 18 hours 0 ng mL TNFa 1 ng mL TNFa 10 ng mL TNFa 100 ng mL TNFa pGreenFire1 NFxB Luciferase Assay 293TN cells were transduced with either pGF1 NFKB or the empty vector pGF1 mCMV and treated with the indicated amount of TNFa for 18 hours B pGFI NFk amp IB pGF1 mCMV ng mL TNFa Fig 2 The top panel shows the results of an NF KB reporter cell line created by infection with the pGreenFire1 NF kB reporter virus Transduced cells were plated in 12 well dishes and then treated with the indicated amounts of TNF a GFP fluorescence was assessed by fluorescence microscopy The bottom panel shows the results of a luciferase assay performed on the 293TN cel
29. uction with SBI s PathNet pGreenFire1 vectors or with constructs made in any of SBI s FIV or HIV based lentivectors D Technical Support For more information about SBI products and to download manuals in PDF format please visit our web site http www systembio com For additional information or technical assistance please call or email us at 888 266 5066 Toll Free 650 968 2200 outside US Page 15 Page 16 System Biosciences SBI User Manual System Biosciences SBI 265 North Whisman Rd Mountain View CA 94043 Phone 650 968 2200 888 266 5066 Toll Free Fax 650 968 2277 E mail General Information info systembio com Technical Support tech systembio com Ordering Information orders systembio com ver 3 140110 www systembio com
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