Retroviral Expression Systems - Applied Biological Materials
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1. Alternatively recombinant retrovirus can also be easily purified using an ion exchange based filter aom Cat G130 Page 11 of 20 Retroviral Expression Handbook Protocol G Determining Viral Titre It is useful to titre viral supernatants before proceeding with transduc tion experiments for the following reasons Ensure that the viral stock is viable Determine the percentage of target cells that can be transduced with pseudoviral stock Control the number of integrated viral constructs per target cell The simplest protocol for measuring titres uses a positive control expres sion plasmid i e GFP mixed with expression construct as an internal control at a ratio of 1 100 and is packaged into pseudoviral particles In an alterna tive approach the GFP control plasmid can be packaged separately but in parallel with your construct as an external control In this scenario the control plasmid can be used to check and optimize the transfection packaging steps To determine the relative viral titre transduce a target cell line such as MDA MB 468 in the presence of Polybrene 2Ug ml for 12 16 hours Count the number of GFP expressing cells by either fluorescence microscopy or FACS 1 For each viral stock plate MDA MB 468 cells one day prior to viral infection in a 24 well plate at a density of 0 6 1x10 cells per well Add Iml of complete DMEM with serum and antibiotics and incubate cells at 37 C with 5 CO ov2. technical abmGood com SIRNA sIRNA abmGood com Suite 8 13520 Crestwood Place Richmond BC Canada Distributors North America Canada Applied Biological Materials Inc Tel 604 247 2416 1 866 757 2414 Fax 604 247 2414 www dbomGood com International Australia Biosensis Pty Ltd Tel 61 43 166 5519 Email sales biosensis com www biosensis com Germany BioCat GmbH Tel 49 0 6221 714 1516 Email info biocat com www biocat com Italy MICROTECH s r l Tel 39 081 6107435 Email microtech microtech eu www microtech eu South Korea CMI Biotech Tel 02 444 7101 Email cmibio cmibio com www cmibio com V6V 2G2 Business Development United States Applied Biological Materials Inc Tel 604 247 2416 1 866 757 2414 Fax 604 247 2414 www abmGood com Belgium Gentaur Tel 32 2 732 5688 Email ea gentaur com www gentaur com India G Biosciences Tel 0120 432 3330 Email rohit gbiosciences com www GBiosciences com Japan Cosmo Bio Co Ltd Tel 03 5632 9610 9620 Email mail cosmobio co jp www cosmobio co jp Taiwan Interlab Co Ltd Tel 886 2 2736 7 100 Email service internab com tw www interlab com tw Retroviral Expression Handbook bd abmGood com Mexico Quimica Lavoisier S A de C V Tel 52 333 848 8484 Email informes lavoisier com mx www lavoisier com mx France Gentaur Tel 01 43 25 01 50 Email ea gentaur com www gentaur com I
3. Hpal 5 GGA ATT CGG GCC CAG ATC TAT CGA TGT TAA Not Sall SnaBl Swal CGC OGC CGC GTC GAC TAC GTA ATT TAA ATT ACC PES 5 ae ener eee ee _ si BamHI CAT ACG ACG TCC CAG ACT ACT GAC TCG AGG ATC CGG 3 SV40 prom pRetro E1 HA 5948bp rep origin Baill Barmy EcoRI Himali 5 TCA GAT CTG GGC CCG GAT CCG AAT TCA AGC Hpal Not Swal Mhol TTG TTA ACA GCG GCC GCC ATT TAA ATC TCG AGC 6x His Cial ATC ATC ACC ATC ACC ATT GAA TCG ATA 3 pRetro E2 His 6275bp Figure 3 Map of pRetro HA amp pRetro His Page 5 of 20 Retroviral Expression Handbook Retroviral Expression System Recombinant Retrovirus is a widely used vessel for successful stable expression of any transduced dividing cell type Mann et al 1983 Miller amp Buttimore 1986 During cell division the nuclear mem brane is disintegrated and the viral DNA can access the host genome Retroviruses can integrate into the host genome efficiently giving rise to permanent and stable gene expression Because recombinant ret roviral vectors cannot actively pass through the nuclear membrane the transduction efficiency of target cells with retroviral vectors is low especially in slow dividing primary cells One of the most significant developments in recombinant retro viral technology is the optimization of high titre retrovirus production by transient transfection using 293 derived packaging cell lines Pear W S ef al 1993 This eliminate
4. M Seidman J G Smith J A amp Struhl K Eds 1995 Current Protocols in Molecular Biology John Wiley amp Sons NY Burns J C Friedmann T Driever W Burrascano M amp Yee J K 1993 Vesicular stomatitis virus G glycoprotein pseudotyped retroviral vec tors concentration to very high titer and efficient gene transfer into mam malian and nonmammalian cells Proc Natl Acad Sci USA 90 8033 8037 Coffin J M amp Varmus H E Ed 1996 Retroviruses Cold Spring Harbor Laboratory Press NY Devroe E amp Silver P A 2002 Retrovirus delivered SIRNA BMC Biotechnol 2 1 15 Emi N Friedmann T amp Yee J K 1991 Pseudotyped formation of murine leukemia virus with G protein of vesicular stomatitis virus J Virol 65 1202 1207 Freshney R I 2000 Culture of Animal Cells Fourth Edition Wiley Liss NY Kozak M 1987 At least six nucleotides proceeding the AUG initiator co don enhances translation in mammalian cells J Mol Biol 196 947 950 Mann R Mulligan R C amp Baltimore D 1983 Construction of a ret rovirus packaging mutant and Miller A D amp Buttimore C 1986 Redesign of retrovirus packaging cell lines to avoid recombination leading to helper virus production Mol Cell Biol 6 8 2895 2902 Pear W S Nolan G P Scott M L amp Baltimore D 1993 Production of hightiter helper free retroviruses by transient transfection Proc Natl Acad
5. No G062 e Trypsin EDTA Trypsin Sigma Cat No 13924 e Dulbecco s phosphate buffered saline DPBS VWR Cat No 82020 066 e BD Biocoat Collagen Type 12 well plates BD Biosciences Cat Nos 354500 amp 356500 e Cloning cylinders PGC Scientific Cat No 62 6150 40 45 e NIH 3T3 cells ATCC Cat No CRL 1658 e Calciumfectin abm Cat G099 e Chloroquine Sigma Cat C6628 e Tissue culture plates and flasks Storage e Pack Easy cells in Liquid Nitrogen e All other components at 20 C e Spin briefly to recover contents and avoid repeated freeze thaw cycles Page 7 of 20 Retroviral Expression Handbook Protocol NOTE The following protocol is broken into sections for convenience However time should be taken to read through the full procedure before attempting A Expression Vector Construction Use standard molecular biology techniques to subclone your gene of interest into any one of our retroviral expression vectors Sambrook amp Russell 2001 1 The gene insert should contain an ATG start codon Also adding a Kozak consensus ribosome binding site may improve expression levels in mammalian cells Kozak 1987 Please note that all genes subcloned into a retroviral vector must not intefere with the retroviral life cycle and allow complete transcription of the full length viralgenome Sequences such as poly A signals must not be included as all retroviral vectors already have a poly A signal seq
6. WN 2 gt Protocol 4 5 Six to eight hours later repeat the infection for overnight infec tion second hit The following day split the cells 1 3 to 1 5 if necessary depend ing on the growth rate of cells Incubate in complete DMEM for an ad ditional 24 48 hours Count the fraction of fluorescent cells using FACS analysis You may also count the GFP positive cells under a fluorescent microscope but the results may be less accurate due to inconsistencies in counting Use the average of the fraction of GFP cells in 5 10 random fields to estimate the overall percentage of GFP cells on the plate Calculate viral titre based on the percentage of GFP positive cells over total num ber of cells analyzed by either microscopy or FACS H Alternative Viral Titre Method Traditionally recombinant retroviral titre has been assayed using the number of selection marker resistant colonies following transduction of NIH3T3 cells 1 Plate NIH3T3 cells 24 hours prior to target cell transduction in 6 well plates at a density of 0 5 1x10 cells per well Prepare 20ml of complete media and add 300ul of 0 8mg ml Polybrene Collect virus containing media from packaging cells Filter media through a 0 45um cellulose acetate or polysulfonic low protein binding filter Prepare six 10 fold serial dilutions as follows a Add 1 35ml of the media to six 1 5ml microcentrifuge tubes b Add 150 ul of virus containing
7. a sche matic overview of retrovirus production by transient transfection in the Pack Easy packaging cell line Retroviral Expression Handbook Page 6 of 20 am Q a 7a ep pa Q x lt LLJ VY t gt cD aa JJ M N m Pas O M WN 2 ie Materials Table Retroviral Vectors and Kits Kit Cat No Component Cat No Quantity E512 E513 E518 E519 oRetro E1 Vector E514 10ug 7 oRetro E2 Vector EIS 10ug Jf ORetro HA Vector E516 10ug pRetro His Vector E517 10ug vi Retro Combo Mix E510 1ORxns y vi J vi Pack Easy Cells E511 l Flask y vi vi v Additional Materials Required The following materials and reagents are required but not provided e Dulbecco s Modified Eagle s Medium Invitrogen Cat 11995 e Fetal bovine serum FBS Cat No TM999 500 Note does not need to be heat inactivated e 200 MM L Glutamine Sigma Cat No G7513 e Solution of 10 000 units ml Penicillin G sodium and 10 000 ug ml Strepto mycin sulphate Sigma Cat No P0781 e Complete Medium DMEM supplemented with 100 units ml penicillin G sodium 100 ug ml streptomycin and 10 fetal bovine serum FBS e G418 Cat No C020 Note Make a 10 mg ml active stock solution by dis solving 1g of powder in approximately 70ml of complete medium without supplements Sterile filter and store at 4 C e Puromycin Cat No C021 e Polybrene Hexadimethrine Bromide Cat
8. and should not be construed as a commitment by abm Inc or its affiliated corporations In no event shall abm Inc or its affillated corporations be liable for incidental or consequen tial damages in connection with or arising from the use of this manual and product abm Inc products are warranted to meet our QC testing standards at the time of shipment Notice of problematic products must be made to abm Inc within 15 days of receipt of the product This product warranty limits abm Inc s liability to the replacement of the product only Technical Support For more information on abm Inc products please visit our website htto www abmGood com For additional information or technical assistance please call or email us at Applied Biological Materials Inc Phone 604 247 2416 1 866 757 2414 Fax 604 247 2414 E mail technical abmGood com Page 1 of 20 Retroviral Expression Handbook Biosafety Our Retro Easy expression vector has been specifically designed to have no genetic overlap with any of the packaging plasmids thus eliminates the possibility of generating replication competent retroviruses However as the protocols presented here involve producing handling and storing recom binant retroviruses proficient understanding of safe laboratory practices and potential retroviral hazards is essential Wild tyoe MMLV does not naturally in fect human cells however recombinant retroviral vectors packaged wit
9. by plating cells at several dif ferent densities in the presence of a constant amount of drug If cells are plated at too high a density they will reach confluency before the selection takes effect Optimal plating density is dependent on popu lation doubling time and cell surface area For example cells that dou ble rapidly have a lower optimal plating density than cells that double slowly a Plate cells at several different densities in each of six 10cm tissue culture dishes containing 10ml of the appropriate selective me dium Suggested densities cells 10 cm dish 5x10 1x10 5x10 2x105 1x105 and 5x104 b Incubate the cells for 5 14 days replacing the selection me dia every four days c Examine the dishes for viable cells every two days For selecting stable transfectants use a plating density that allows the cells to reach 80 confluency before massive cell death begins at about day 7 This is the cell density at which cells should be plated for selection of transduced target cells Retroviral Expression Handbook Page 18 of 20 Notes Page 19 of 20 Retroviral Expression Handbook Contact Information Applied Biological Materials Inc Website www domGood com Phone 8 30am 4 300m PST M F Toll Free 1 866 757 2414 Local 604 247 2416 Fax 604 247 2414 24Hr Address Email General Information info abmGood com Order Products order abmGood com Technical Support
10. media to the first tube Mix c Transfer 150ul of viral stock dilution from tube 1 to tube 2 Continue serial dilutions by transferring 150ul of each successive dilu tion to the next prepared tube Transduce NIH3T3 cells by adding Iml of the diluted virus me dium from Step 5 to the wells Final polyorene concentration will be 4ug ml in 3ml Page 13 of 20 Retroviral Expression Handbook Protocol 7 8 The following day change the media Start antibiotic selection 48 hours after infection using appropri ate antibotic concentration based on the killing curve information see Appendix A I Target Cell Transduction The following protocols are general guidelines for the transduction of adherent cells such as NIH3T3 or HeLa It is recommended that users optimize for efficient gene transduction of suspension cells using the same principle as optimizing the transduction of adherent cells Note Multiple rounds of infec tion can improve your results by increasing the number of infected cells as well as increasing the copy number per cell 1 Plate the target cells 12 18 hr before infection at a cell density of 5x104 per 6 well plate Note Retroviral particles can only passively enter the nuclei of actively dividing cells not non dividing cells Add virus to target cells Unless you have determined the viral titre use as Much virus containing medium as possible for the infection Store the remaining viral supernat
11. Sci USA 90 18 8392 8396 Sambrook J amp Russell D W 2001 Molecular Cloning A Laboratory Manual Cold Spring Harbor Laboratory Press Cold Spring Harbor NY Page 17 of 20 Retroviral Expression Handbook Appendix A Titration of Antibiotic Selection Prior to using G418 or Puromycin to generate stable cell lines of trans duced target cells if is important to titrate selection antibiotics to determine the optimal concentration used for the selection of transduced target cells For optimal selection we recommend that you perform two experiments for each drug 1 a titration to determine the optimal drug concentration and 2 an experiment to determine the optimal plating density 1 Titrate at fixed cell density that is going to be used during trans duction of target cells interested a Plate 2x10 cells in each of six 10cm tissue culture dishes containing 10ml of the appropriate complete medium plus varying amounts 0 50 100 200 400 800 100ug ml For Puromycin add the drug at O 0 5 1 2 4 6 8 and 10ug ml b Incubate the cells for 10 14 days replacing the selection me dia every four days or more often if necessary c Examine the dishes for viable cells every two days In gener al use the lowest concentration that begins to give massive cell death in 7 days and kills all the cells within two weeks Once you have determined the optimal drug concentration determine the optimal plating density
12. ant at 80 C As a general guidline use 2ml of viral supernatant for a 6 well size culture vessel in the pres ence of 2 20 Ug ml Polybrene first hit for 8 10 hours and then second hit for overnight Make Polybrene stock at 0 8mg ml Use 20ul well in a 6 well plate and 100ul 10cm dish Notes Titre will decrease 20 40 per freeze thaw cycle based on our in house data The optimal final concentration of polybrene may need fo be empirically determined but it is generally within a range of 2 l0ug ml Excessive exposure to polybrene gt 24hr can be toxic to cells Replace medium with fresh complete medium the follow ing day Some target cells especially primary cells are very sensi tive to conditioned viral supernatant from packaging cells partly due to nutrient starvation This can be alleviated by the following steps a Dilute virus containing supernatant at least 2 fold with fresh media b Expose target cells to the virus for 4 6 hours and then replace with fresh medium though this will significantly decrease transduction efficiency Retroviral Expression Handbook Page 14 of 20 Oo 7a iT ap i Q x lt LLI 7a gt paa ce Cc Protocol 4 The following day depending on the growth rate of cells split the cells 1 3 to 1 5 in the presence of the appropriate selection marker G418 or Puromycin as determined by the killing curve see Appendix A Change medium every 3 4 days to mai
13. aom Retroviral Expression Systems pRetro E1 Vector pRetro E2 Vector pRetro HA Vector pRetro His Vector Retro E1 Expression Kit Retro E2 Expression Kit Retro HA Expression Kit Retro His Expression Kit E514 E515 E516 E517 E512 E513 E518 E519 www abmGood com WOD pOOHWge MMM Table of Contents Notice to the Purchaser Technical Support Biosafety Protocol at a Glance Retroviral Expression System Materials Additional Materials Required Storage Protocol Expression Vector Construction Culturing Pack Easy Cells Subculturing Pack Easy Cells 293T Cells Viral Production Concentrating Virus Determing Viral Titre Alternative Viral Titre Method Target Cell Transduction Troubleshooting References Appendix A Titration of Antibiotic Selection Notes Contact Information Retroviral Expression Handbook 16 17 18 19 20 Notice to the Purchaser The products are for research use only Use in and or for diag nostics or therapeutics is strictly prohibited By opening and using the product the purchaser agrees to the following The components in this kit may not be distributed resold or modified for resale without prior written approval from Applied Biological Materials abm Inc If you do not agree to the above conditions please return the UNOPENED product to abm Inc within ten 10 days of receipt for a full refund The information in this docu ment is subject to change without notice
14. ernight Note If is possible to use bigger culture dishes for transduction especially when a large number of cells is required for FACS analysis In this case the amount of cells should be adjusted depending on the growth area of the well plate 2 On the following day prepare complete DMEM media with 10 FBS and Polybrene to a final concentration of 2ug ml Note Polybrene is a polycation that neutralizes charge interactions to increase binding be tween 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 10ug ml Excessive ex posure to Polybrene gt 12 hr can be toxic to some cells 3 Remove culture media and replace with 0 5ml of complete DMEM with 10 serum and Polybrene from Step 2 For each viral stock use three wells Infect MDA MB 468 cells with 100ul 500ul 1 0ml and 2 0m of viral supernatant in the presence of 2ug ml Polybrene in the early morning Additional 1 9ml 1 5ml and 1 0ml of complete media should be added to viral supernatant that are less than 2 0ml to bring the total infection volume to 2 0ml For control wells add 2ml of DMEM medium with Polybrene Incubate cells at 37 C with 5 CO for 6 8 hours Retroviral Expression Handbook Page 12 of 20 Oo 7a 7a ap Q x lt LLJ Ta gt gt ie 2 JJ D at eo a V m Pas O D
15. fter any spills of viable material e All cultures stocks and other regulated wastes are decontaminated be fore disposal by an approved decontamination method like autoclaving Retroviral Expression Handbook Page 2 of 20 Protocol at a Glance Plate Packaging Cells Expression Vector 5 3 10hr Target Cell Infection gt Transfect Plate Targeting Cells 18hr 48 72hr l oe Infect With Virus Collect Virus R 1 Week 2 3 Days Select Cells and Analyze Determine Viral Titre Figure 1 Overview of transient retrovirus production To produce recom binant retrovirus both retroviral expression vector and packaging mix are co transfected into packaging Pack Easy cells After 48 72 hours viral supernatant is collected for either titre determination or transduction of target cells Page 3 of 20 Retroviral Expression Handbook EcoRI Apal Bglll Clal Hpal 5 GGA ATT CGG GCC CAG ATC TAT CGA TGT TAA Not Sall SnaBl Swal Xhol CGC GGC CGC GTC GAC TAC GTA ATT TAA ATC TCG BamHI AGG ATC CGG 3 SV40 prom pRetro E1 5921bp rep origin Baqlll hol Apal BamHI Hpal 5 CAG ATC TCG AGG GGC CCG GAT CCG TTA ACG Sphl Swear Hindi EcoRI CAT GCA TTT AAA TAA GCT TCG AAT TCG TTA GGC Sal Not CAT TAA GGC CTG TCG ACA AGC GGC CGC CTC GGC Clot CAA ACA TCG ATA 3 pRetro E2 6294bp Figure 2 Map of pRetro El amp PRetro E2 Retroviral Expression Handbook Page 4 of 20 EcoRI Apai Baill Clai
16. h VSV G envelope are capable of infecting human cells In addition depending on your gene insert the viral supernatants produced by co transient transfection could be potentially hazardous For these reasons extra precautions should be observed when working with recombinant retroviruses expressing known oncogenes or other toxic proteins General guidelines in most jurisdictions require that retroviral pro duction and transduction be performed in a Biosafety Level 2 BL2 facility For more information about the BL 2 guidelines and retrovirus handling refer to Biosafety in Microbiological and Biomedical Laboratories 5th Edition published by the Center for Disease Control CDC This document may be downloaded at the following address htto www cdc gov biosafety publications ombl5 index htm Additional information is also available at httpo ombl od nih gov contents htm All published BL 2 guidelines for proper waste decontamination should be strictly followed It is also important to consult with the health and safety officer s at your institution for guidelines regarding the use of retroviruses and to always follow standard microbiological practices which include e Wear gloves and a lab coat at all times e Always work with pseudoviral particles in a Class Il culture facility and that all procedures are performed carefully to minimize splashes and aerosols e Work surfaces are decontaminated at least once a day and a
17. ion efficiency Virus harvested too early Vector too large Polybrene not used Improper selection dur ing titration Viral stocks stored incorrectly Avoid freeze thaw viral supernatant each cy cle decreases titre approx 20 40 Check for presence of poly A between LTR s Optimize transfection Harvest virus 48 72hrs after transfection The limit of the vector is 8 3kb from LTR to LTR Add Polybrene during titration amp transduction Perform an antibiotic kill Curve on titration tar gets prior to titration Determine antibiotic sensitivity of target cells by performing a killing curve Use minimum antibi otic concentration required Poor Infection Target cells not dividing Plate cells at lower confluency activate with Improper Polybrene concentration Efficiency mitogen or use another method to induce cell division Low titre See previous section Target Cell Virus supernatant may be Dilute viral media or shorten exposure time to Viability Poor affecting cell growth viral supernatant Titrate Polybrene or shortern exposure time to viral supernatant Low Expression Level Possible promoter inactivation Poor cell viability Retroviral Expression Handbook Split cells activate with mitogen treat cells with 5 azacytidine or choose a tissue specific pro moter Check growth parameters Page 16 of 20 References Ausubel F M Brent R Kingdom R E Moore D
18. ntain consistent selection drug concentration The selection should be complete in 2 weeks for most target cell types JJ gt ot N m Pas O gt WN 2 fe 5 Pick up 10 20 colonies for expansion and screening for stable clones with high level of transgene expression Page 15 of 20 Retroviral Expression Handbook Troubleshooting Problem Possible Cause Solution No Colony Wrong antibiotic or antibiotic Use kanamycin at 50ug ml of LB agar media Growth concentration too high Poor transformation efficiency Check transformation efficiency using control plasmid like a parental plasmid Low Plasmid Retroviral constructs use Grow more liquid culture and purify using low Yield a low copy PBR322 ori copy purification procedures Plasmid Difficult to Grow Plasmid may rearrange due to presence of LTR s Switch to alternate E coli strain for unstable DNA selection Poor Viability Improper thawing procedures Improper culture media Follow thawing procedures in Culturing Pack Easy Cells section Grow Pack Easy cells in DMEM with 10 FBS use high glucose DMEM media only Poor Transfec tion Efficiency Cell density not optimal a cell density of 85 90 is best for high titre virus production Optimize DNA and transfection amounts and exposure time reagent Low Titre lt 10 cfu ml Too many freeze thaw cycles Truncated viral RNA Poor transfect
19. ock of chloroquine in distilled water and sterile filter l 2 hours before transfecton replace media with media containing 25uM of chloroquine Pear et al 1993 For each transfection prepare Solution A and Solution B in sepa rate 5ml sterile tubes Solution A Add components in the following order and mix well 10 15ug retroviral vector DNA 100ul 1Oug Retro Combo Mix 64ul 2M CaCl Xul H O Top up to 500u l Total Solution B 500ul 2X HBS a Carefully and slowly vortex Solution B while adding Solution A drop wise Alternatively blow bubbles into Solution B with an autopi pettor while adding Solution A drop wise b Incubate reaction at room temperature for 10 15 minutes c Add mixture drop wise to the culture plate gently but quickly d Incubate the plates at 37 C for 5 8 hours without disturbing e Take out transfection complex and add 10ml of fresh media f Incubate overnight at 37 C Retroviral Expression Handbook Page 10 of 20 Oo 7a 7a ap pan Q x lt LLI Ta gt pa e9 o JJ M ot V m Pas O gt V a eo Protocol 4 The next day change media and incubate for another 24 hours Plate target cells at 50 60 confluency in 6 well plates to prepare for transduction the following day Harvest Pack Easy cell supernatant Feed cells with another 10ml fresh media and incubate for another 24 hours Filter viral superna
20. s the time consuming process of gen erating retrovirus producer cell lines In addition the procedure signifi cantly increases the safety of the recombinant retrovirus production as it eliminates the culturing of stable virus producing cell lines abm s retroviral expression systems provide a simple and effi cient means for producting high titre recombinant retroviruses All of our retroviral vectors contain user friendly multiple cloning sites MCS allowing for easy cloning of your gene of interest into our vectors In addition our PRetro HA and pRetro His vectors offer the flexibility of adding either HA or His tags in frame to your gene of interest Our Retro Combo Mix contains all the genetic elements re quired for the production of high titre retroviruses using the transient transfection approach The Gag Poly and VSV G Envelope plasmids of the Retro Combo Mix have been meticulously optimized for high titre retroviral production The Retro Combo Mix is compatible with retroviral vectors derived from the Murine Stem Cell Virus MSCV Moloney murine leukemia virus MOMULV and the Moloney murine sarcoma virus MOMUSV With Retro Combo packaging mix virions are pseudotyped with the envelope glycoprotein from the vesicular stomatitis virus VSV G VSV G mediates viral entry through lipid bind ing and plasma membrane fusion giving rise to higher transduction efficiency Burns et al 1993 Emi et al 1991 Figure 1 shows
21. srael BioConsult Tel 972 0 2 566 7043 Email sales bioconsult co il www bioconsult co il Singapore Bio REV PTE Tel 65 6273 3022 Email allan bio rev com bio rev com United Kingdom NBS Biologicals Ltd Tel 44 0 1480 433875 Email info nbsbio co uk www nbsbio co Uuk Page 20 of 20 The Source for any Antibody siRNA and Viral Vector The Depot for and Transfection Reagents r PCR qPCR
22. sy cells do not adhere well to culture vessels and extra care should be taken fo avoid dislodging the cells during media change over When changing the medium during retrovirus production add the me dium slowly against the side of culture vessels JJ 9 ot n N m Pas O a WN ae ma Split the cells as follows 1 Aspirate and wash cells once with serum free culture medium 2 Add 4 6ml of trypsin EDTA solution and incubate at room tem perature for 2 5 minutes Add 3 ml of complete media to inhibit trypsinization Resuspend cells gently by pipetting Transfer cells into a 15ml culture tube 5 Spin at 1000g for 5 minutes to pellet cells Aspirate the supernatant then add 2ml of complete media 7 Resuspend cells by pipetting then plate cells at a density of 80 for virus production on the following day D 293T Cells The human 293T cell line is widely used for optimal retrovirus produc tion Naldini et al 1996 The health of 293T cells at the time of transfection is a critical factor for the success of retrovirus production The use of un healthy cells will negatively affect the transfection efficiency resulting in low titre retroviral stocks For optimal retrovirus production follow the guidelines below to culture 293T cells before use in transfection e Ensure cell viability is greater than 90 e Subculture and maintain cells in complete medium containing 0 1mM MEM Non Essen
23. tant through a 0 45um filter to remove cell de bris Note The 0 45uM filter should be cellulose acetate or polysulfonic filter low protein binding Do not use a nitrocellulose filter because it binds to proteins in the retroviral membrane and destroys the virus Either infect target cells as shown in the Target Cell Transduction section or freeze down viral supernatant at 80 C for future applications Note Aliquot filtered supernatant into single use tubes to avoid multi ple freeze thaw cycles Multiple freeze thaw cycles should be avoided since titre can drop as much as 20 40 with each cycle Higashikawa amp Chang 2001 Kwon et al 2003 Store tubes at 80 C and no cryoprotec tant is required Collect the second viral supernatant after the 24 hours Filter through 0 45um a filter and either infect target cells or freeze viral super natant down at 80 C for future applications F Concentrating Virus If high titre virus is needed the retroviruses can be concentrated by ultracentrifugation 1 Remove cell debris and aggregated virus by centrifugation at 2000g for 5 minutes at 4 C Pellet the virus at 50 000g for 90min at 4 C Remove the su pernatant Resuspend the virus to 0 5 1 of the original volume in vi ral suspension buffer 50mM Tris HCI pH 7 8 130MM NaCl 1mM EDTA Determine the viral titre of the pre and post concentrat ed viral supernatants using the protocol discussed in the follow ing section
24. tial Amino Acids 4mM L Glutamine 1mM sodium pyru vate e 500ug ml Geneticin and 10 FBS e Do not allow cells to overgrow before passaging e Use cells that have been subcultured for less than 16 passages Page 9 of 20 Retroviral Expression Handbook Protocol E Viral Production Recombinant retroviruses are produced in Pack Easy cells through transient transfection which can be done by standard Calcium Phosphate reagents Cat No G099 For reasons still unknown retrovirus production with standard calcium phosphate transfections consistently produces higher titre 1 5x than those with other types of transfection reagents like Lipofectamine Transfections are generally performed in 10cm dishes Higher titre retroviruses can be produced when Pack Easy cells are at 85 to 95 confluency As a guideline subculturing a confluent 10cm culture plate in 1 2 ratio or a con fluent 15cm dish to five 10cm dishes 12 16 hours before transfection provide suitable cell density for transfection the following day 1 One day before transfection plate Pack Easy cells in a 10cm tissue Culture plate so that they will be 90 95 confluent on the day of transfection i e 5x10 cells in 1Oml of growth medium with serum Prior to transfection add 10ml media DMEM with 10 FBS con taining 25uM chloroquine final concentration Note Adding chloro quine prior to transfection may increase transfection efficiency 2 3 fold Prepare a 25mM st
25. uence following their 3 LTR Coffin ef al 1996 2 Digest both the insert gene and the vector with the appropriate restriction enzyme s followed by purification Ligate the insert gene fragment into the vector Transformligation productsintoE colicells Screen for recombinant plasmids by restriction analysis and con firm by sequencing B Culturing Pack Easy Cells The Pack Easy Cell Line is widely used for optimal retrovirus production due fo Its high transfection efficiency The health of Pack Easy cells at the time of transfection Is a critical factor for the success of retrovirus production The use of unhealthy cells will negatively affect the transfection efficiency resulting in low titre retroviral stocks For optimal retrovirus production follow the guidelines below to culture Pack Easy cells before transfection Make sure the cells possess greater than 90 viability Subculture and maintain cells in complete medium containing O 1m M MEM Non Essential Amino Acids 4mM L Glutamine 1mM sodium pyruvate 500ug m Geneticin and 10 FBS Donot allow cells to overgrow before passaging Retroviral Expression Handbook Page 8 of 20 Oo 7a 7a ap Q x lt LLJ a gt i o Protocol C Subculturing Pack Easy Cells In general Pack Easy cells should be plated at 10 cells per 10cm plate and split every 2 3 days when they reach 90 confluency Note Pack Ea
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