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Retroviral Gene Transfer and Expression User Manual

1. Recommended Concentration pg ml Cat No Antibiotic Selecting Colonies Maintenance 631308 G418 5 g 100 800 200 631307 G418 1 g 631306 Puromycin 100 mg 0 25 10 0 25 631305 Puromycin 25 mg 631309 Hygromycin B 1 g 50 400 100 1 When selecting for single colonies the appropriate dose must be determined empirically for your specific cell line Test a dosage range using dishes of untransfected cells and choose the dose that kills all of the cells in 3 5 days If all the cells die in less than 24 hr you should use a lower dose G Competent E coli Cells Suitable for Retroviral Vector Manipulations Retroviral and lentiviral vector possess long terminal repeats that can make them unstable in some E coli strains For manipulations of these vectors we recommend Stellar Competent Cells Cat No Product Size 636763 Stellar Competent Cells 10 x 100 ul 636766 Stellar Competent Cells 50 x 100 ul 636765 Stellar Electrocompetent Cells 10 transformations H Plasmid Purification Transfection Grade Cat No Product Size 740412 10 NucleoBond Xtra Midi Plus 10 preps 740416 10 NucleoBond Xtra Maxi Plus 10 preps 740422 10 NucleoBond Xtra Midi EF Plus 10 preps 740426 10 NucleoBond Xtra Maxi EF Plus 10 preps l In Fusion HD Cloning Plus In Fusion is a revolutionary technology that greatly simplifies cloning For more information visit www clontech com infusion Cat No In F
2. 2 x 10 cells ml EcoPack 2 293 Cell Line Cat No 631507 e im EcoPack 2 293 Cell Line 2 x 10 cells ml AmphoPack 293 Cell Line Cat No 631505 e imi AmphoPack 293 Cell Line 2 x 10 cells ml Additional Materials Required A Mammalian Cell Culture Supplies e Packaging Cell Line growth medium 90 Dulbecco s Modified Eagle s Medium DMEM with high glucose 4 5 g L 10 Fetal Bovine Serum 4 mM L glutamine and 3 7 g L sodium bicarbonate Sigma Aldrich Co No D5796 Add 1 mM sodium pyruvate e Cell growth medium and supplies specific for your target cells e Sodium pyruvate solution 100 mM sterile filtered Sigma Aldrich Co Cat No 8636 for supplementing cell culture media e Penicillin streptomycin solution of 10 000 units ml penicillin G sodium and 10 000 ug ml streptomycin sulfate 100X Sigma Aldrich Co Cat No P0781 e Trypsin EDTA Trypsin Sigma Aldrich Co Cat No T3924 e Dulbecco s phosphate buffered saline DPBS Sigma Aldrich Co Cat No D8662 e L glutamine solution 200 mM sterile filtered Sigma Aldrich Co Cat No G7513 Optional e Cell Freezing Medium with or without DMSO Sigma Aldrich Co Cat No C6164 or No C6039 e Tissue culture plates 100 mm for packaging cell transfections other plates and flasks as required e 15 ml polystyrene conical centrifuge tube for the CalPhos transfection protocol e g BD Biosciences Cat No 352099 e Sterile microfuge tubes 1 5 ml for u
3. 800 662 2566 toll free Telephone 800 662 2566 toll free Fax 800 424 1350 toll free Fax 800 424 1350 toll free Web www clontech com Web www clontech com E mail orders clontech com E mail tech clontech com Notice to Purchaser Clontech products are to be used for research purposes only They may not be used for any other purpose including but not limited to use in drugs in vitro diagnostic purposes therapeutics or in humans Clontech products may not be transferred to third parties resold modified for resale or used to manufacture commercial products or to provide a service to third parties without written approval of Clontech Laboratories Inc Your use of this product is subject to compliance with any applicable licensing requirements described on the product s web page at www clontech com It is your responsibility to review understand and adhere to any restrictions imposed by such statements RetroNectin is a trademark of Takara Bio Inc SYBR is a registered trademark of Molecular Probes Inc Clontech the Clontech logo CalPhos EcoPack In Fusion Knockout Lenti X Living Colors ProteoTuner RetroPack Retro X SMART Stellar Tet Express Tet On and Xfect are trademarks of Clontech Laboratories Inc All other marks are the property of their respective owners Certain trademarks may not be registered in all jurisdictions Clontech is a Takara Bio Company 2013 Clontech Laboratories Inc This document has
4. Figure 4 Schematic for titering retrovirus supernatants with the Retro X qRT PCR Titration Kit Flow cytometry For Retro X vectors containing a fluorescent marker cells can be transduced using the protocol in Section VII B followed by counting the cells 24 48 hr later using fluorescence and flow cytometry Titers determined in this manner are generally higher than those determined by antibiotic selection Antibiotic selection For Retro X vectors that contain a selectable marker cells are infected with serial dilutions of the virus stock and then selected for stable transductants using the appropriate antibiotic Titers are calculated from the number of drug resistant colonies that develop after selection is completed Section VIILB www clontech com Clontech Laboratories Inc A Takara Bio Company Page 25 of 31 Retroviral Gene Transfer and Expression User Manual 061113 B Protocol Determining Viral Titer Using Antibiotic Selection NOTE This protocol can be completed in 7 14 days 1 Plate HT 1080 cells or NIH 3T3 if you are using ecotropic virus in one 6 well plate the day before performing the titration infections Plate 2 x 10 cells well in 2 ml of medium Reserve at least one well for a no infection control NOTE You can use other cell lines to determine viral titer but HT 1080 cells are widely accepted as the standard target cell for titrating retrovirus NIH 3T3 cells for ecotropic virus because these ce
5. C1 Cat Nos 632567 amp 632568 and pRetroQ DsRed Monomer N1 C1 Cat Nos 632507 amp 632508 allow you to fuse your gene of interest to monomeric fluorescent proteins which make ideal fusion tags These vectors are available in both N and C terminal formats b Retroviral Vectors that are Ideal for Reporter Studies pRetroX IRES ZsGreen1 Cat No 632520 and pRetro X IRES DsRedExpress Cat No 632521 provide bright fluorescent proteins that are excellent reporters for gene expression or for measuring transduction transfection efficiency www clontech com Clontech Laboratories Inc A Takara Bio Company Page 4 of 31 Retroviral Gene Transfer and Expression User Manual 4 Tet Inducible Retroviral Vectors Tet On 3G amp Tet Express Systems These retroviral vectors provide tightly controlled expression of your transgene using Clontech s Tet On 3G and Tet Express Inducible Expression Systems 5 061113 a Tet Inducible Expression Using a Retroviral Vector Set The Retro X Tet On 3G Inducible Expression System Cat No 631188 is a tightly regulated tetracycline inducible retroviral gene expression system using Retro X Q Vector technology pRetroX Tet3G and the pRetroX TRE3G to provide efficient retroviral delivery and inducible expression of your gene of interest In the presence of doxycycline Dox the Tet On 3G transactivator protein expressed by pRetroX Tet3G specifically binds and activates high level trans
6. For guaranteed transfection grade plasmid DNA we recommend using NucleoBond Xtra Midi Plus or Maxi Plus Kits Section IIL H For rapid production of endotoxin free transfection grade plasmid DNA use NucleoBond Xtra Midi EF Plus or Maxi EF Plus Kits Section I H Make sure to perform a diagnostic digest to verify the integrity of the recombinant transfer vector after propagation www clontech com Clontech Laboratories Inc A Takara Bio Company Page 17 of 31 Retroviral Gene Transfer and Expression User Manual A gt B 7 New column filter Fast filtration NucleoBond Finalizer k E for fast DNA precipitation g x g E a S Improved t 2 silica material o 3 High binding capacity Low silica 7 resin bed High flow rate Figure 2 Advanced features of NucleoBond Xtra Maxi and Midi Columns and NucleoBond Finalizer NucleoBond Xtra columns contain a high flow column filter that minimizes clogging and clears debris from cell lysates during column loading An improved silica resin provides high DNA binding capacity and a wide column diameter keeps the resin bed low for maximum flow rates Panel A The NucleoBond Finalizer system speeds preparation and increases purity by capturing precipitated DNA on a syringe filter where it can be easily washed and eluted Panel B VI Working with Retroviral Packaging Cell Lines A General Cell Culture and Retrovirus Information The protocols in this User Manual prov
7. The antibiotic concentration which caused massive cell death when determining the appropriate dose via titration could be too high Use a lower antibiotic concentration for selection of stably transfected cell clones Cells were not properly frozen See Section VI C Poor cell viability Cells were not properly thawed See Section VI B 061113 www clontech com Clontech Laboratories Inc A Takara Bio Company Page 30 of 31 Retroviral Gene Transfer and Expression User Manual XII References Ausubel F M Brent R Kingdom R E Moore D M Seidman J G Smith J A amp Struhl K eds 1995 Current Protocols in Molecular Biology John Wiley amp Sons NY Bloor S Maelfait J Krumbach R Beyaert R amp Randow F 2010 Endoplasmic reticulum chaperone gp96 is essential for infection with vesicular stomatitis virus Cell 107 15 6970 6975 Coffin J M Hughes S H amp Varmus H E eds 1997 Retroviruses Cold Spring Harbor Laboratory Press Cold Spring Harbor NY Freshney R I 2010 Culture of Animal Cells A Manual of Basic Technique and Specialized Applications 6th Edition Wiley Liss New York NY Green M R amp Sambrook J 2012 Molecular Cloning A Laboratory Manual Fourth Edition Cold Spring Harbor Laboratory Cold Spring Harbor NY Contact Us For Assistance Customer Service Ordering Technical Support Telephone
8. by approximately 2 4 fold Limit the number of freeze thaws Suboptimal selection procedure during titration Perform an antibiotic kill curve on the cell line prior to using it for titration 061113 www clontech com Clontech Laboratories Inc A Takara Bio Company Page 29 of 31 Retroviral Gene Transfer and Expression User Manual Problem Possible Explanation Solution D Transduction of Target Cells Low titer See Section C or use the Retro X Concentrator I E to increase your available titer up to 100 fold without ultracentrifugation Poor transduction efficiency Low viability of target cells during transduction Optimize culture conditions for target cells prior to infection Packaging cell line conditioned media may affect cell growth dilute viral supernatant or shorten exposure time to viral supernatant Consider using RetroNectin Reagent and the RetroNectin Bound Virus transduction protocol Excessive exposure to Polybrene optimize amount titrate or shorten exposure time to viral supernatant Viral supernatant contains transduction inhibitors Use RetroNectin Reagent or RetroNectin coated plates in the RetroNectin Bound Virus transduction protocol which allows virions to bind the RetroNectin substratum and be washed free of inhibitors prior to target cell infection Division rate of cells not sufficient to permit transduction Stimulate c
9. defining any needed waste decontamination or medical surveillance policies e Safety equipment Biological Safety Cabinet preferably a Class II BSC laminar flow hood with a HEPA microfilter used for all manipulations of agents that cause splashes or aerosols of infectious materials exhaust air is unrecirculated PPE protective laboratory coats gloves face protection as needed e Facilities Autoclave available for waste decontamination Chemical disinfectants available for spills www clontech com Clontech Laboratories Inc A Takara Bio Company Page 16 of 31 Retroviral Gene Transfer and Expression User Manual V Plasmid Vector Manipulations A General Molecular Biology Techniques These protocols contain only general information for propagating plasmid vectors and for preparing your customized expression construct in a Retro X Vector For users requiring more information on standard molecular biology practices and cloning techniques we recommend the following laboratory references Current Protocols in Molecular Biology ed by F M Ausubel et al 1995 John Wiley amp Sons NY Molecular Cloning A Laboratory Manual Fourth Edition ed by Green M R amp Sambrook J 2012 Cold Spring Harbor Laboratory Press NY B Plasmid Vector Propagation amp Construction of Your Customized Retro X Vector 1 061113 To ensure that you have a renewable source of plasmid DNA transform each of the plasm
10. multiplicity of infection MOD it is necessary to titrate your retrovirus stocks Freshly harvested virus stocks can be titrated immediately or frozen in aliquots at 80 C and then titrated Note that each freeze thaw cycle can reduce the functional titer of the virus stock by up to 2 4 fold Titrations are important for determining the relative virus content of stocks prepared from different vectors and for Confirming the viability of virus stocks Determining the optimal transduction conditions Adjusting the MOI to control the viral copy number of transduced cells Determining the maximum number of cells that can be infected by a virus stock Titration can be accomplished using different methods depending on the presence of a selectable or fluorescent marker qRT PCR Clontech offers a convenient Retro X qRT PCR Titration Kit see Section III C for rapid titration of retroviral supernatants It employs One Step qRT PCR and SYBR Green chemistry in a fast 4 hr protocol that can be used with any retroviral vector regardless of the marker involved and is beneficial for comparing the titers of different vectors and for titrating freshly harvested virus stocks Since this titration kit does not rely on antibiotic selection all particles regardless of genome sequence or infectivity can be quantified and compared S y Harvest Viral RNA qRT PCR Data analysis retroviral purification supernatant amp DNase treatment
11. selective antibiotics and 10 DMSO Dispense 1 ml aliquots into sterile cryovials and freeze slowly 1 C per min For this purpose you can place the vials in Nalgene cryo containers Nalgene Cat No 5100 001 and freeze at 80 C overnight Alternatively place vials in a thick walled styrofoam container at 20 C for 1 2 hr Transfer to 80 C and freeze overnight The next day remove the vials from the cryo containers or styrofoam containers and place in liquid nitrogen storage or an ultra low temperature freezer 150 C for storage Two or more weeks later plate a vial of frozen cells to confirm viability D Packaging Cell Line Maintenance and Passaging For optimal results expand your packaging cell line and freeze multiple aliquots to use as master cell stocks as described in Section C Then create working stocks from the master cell stocks periodically replacing your working stock from a fresh master Working stocks that are passaged for extensive periods of time may show reduced performance Cells should be plated at 10 cells per 100 mm dish and split every 2 3 days when they reach 70 80 confluence Do not allow your cells to become overconfluent The doubling time for GP2 293 EcoPack 2 293 and AmphopPack 293 cell lines is 24 36 hr The RetroPack PT67 Cell Line has a very short doubling time of 16 hr www clontech com Clontech Laboratories Inc A Takara Bio Company Page 20 of 31 Retroviral Gene Transfe
12. that allows fast simple and highly efficient concentration of any retroviral stock without using ultracentrifugation In the simple protocol retroviral supernatant is mixed with the Retro X Concentrator reagent incubated for a short period and centrifuged in a standard centrifuge 1 Transfer clarified supernatant Section VII B Step 10 or Section VIIC Step 9 to a sterile container and combine 1 volume of Retro X Concentrator with 3 volumes of clarified supernatant Mix by gentle inversion Larger volumes may be accommodated through the use of larger i e 250 ml or 500 ml centrifuge tubes It is recommended to start with at least 10 ml or more of viral supernantant if the viral titer is expected or known to be low 10 IFU ml NOTE For easy calculation of the amount of Retro X Concentrator to use simply measure the amount of viral supernatant to be concentrated divide by 3 and add the resulting amount of Retro X Concentrator to your viral supernatant Incubate mixture overnight at 4 C Centrifuge sample at 1 500 x g for 45 minutes at 4 C After centrifugation an off white pellet will be visible Carefully remove supernatant taking care not to disturb the pellet Residual supernatant can be removed with either a pipette tip or by brief centrifugation at 1 500 x g Gently resuspend the pellet in 1 10 to 1 100th of the original volume using complete DMEM PBS or TNE The pellet can be somewhat sticky at first but will g
13. the target cells Harvest the cells for analysis or proceed with selection using the appropriate antibiotic NOTE To determine the efficiency of transduction you can subject a small subpopulation of cells to antibiotic treatment and harvest the remaining cells for analysis The cells should be used as soon as possible but not earlier than 24 hr after transduction www clontech com Clontech Laboratories Inc A Takara Bio Company Page 28 of 31 Retroviral Gene Transfer and Expression User Manual XI Troubleshooting Guide Problem Possible Explanation Solution A Vector Cloning Plasmid is difficult to grow or clone Some viral vectors may undergo rearrangements between the 5 and 3 LTRs when propagated in less than optimal E coli host strains Use Stellar Competent Cells Cat No 636763 to produce high DNA yields and to minimize the potential for DNA rearrangements B Packaging Cells Poor viability upon thawing Improper thawing techniques Use thawing procedure in Section VI B Incorrect culture medium Use DMEM with additives listed in Section III A Improper tissue culture plasticware Use collagen l coated plates to aid cell adherence during initial seeding Slow growth Incorrect culture medium Use DMEM with additives listed in Section III A Cells do not attach to plate Improper tissue culture plasticware Use collagen l coated plates to aid cell adherence durin
14. therapy trials attesting to their potential ability to express genes in vivo IMPORTANT For these reasons due caution must be exercised in the production and handling of any recombinant retrovirus The user is strongly advised not to create retroviruses capable of expressing known oncogenes in amphotropic or polytropic host range viruses For more information on Biosafety Level 2 agents and practices download the following reference Biosafety in Microbiological and Biomedical Laboratories BMBL Fifth Edition February 2007 HHS Pub No CDC 93 8395 U S Department of Health and Human Services Centers for Disease Control and Prevention and NIH Available on the web at http www cdc gov biosafety publications bmbl5 BMBL pdf Biosafety Level 2 The following information is a brief description of Biosafety Level 2 It is neither detailed nor complete Details of the practices safety equipment and facilities that combine to produce a Biosafety Level 2 are available in the above publication If possible observe and learn the practices described below from someone who has experience working with retroviruses Summary of Biosafety Level 2 1 Practices Standard microbiological practices Limited access to work area Biohazard warning signs posted Minimize production of aerosols Decontaminate potentially infectious wastes before disposal Use precautions with sharps e g syringes blades Biosafety manual
15. ug envelope plasmid e g pVSV G e AmphoPack 293 EcoPack 2 293 RetroPack PT67 15 ug retroviral plasmid Add sterile H5O to a final volume of 1 250 ul While vortexing at moderate speed add 1 250 ul 2X HEPES Buffered Saline HBS dropwise Incubate each DNA CalPhos mixture for 5 min at room temperature Own BR Add the entire 2 5 ml of DNA CalPhos precipitate dropwise to the cell culture medium from Step 1 Rock the plate gently back and forth to mix NOTE It is not necessary to remove serum from your cell culture medium 7 Incubate the plate at 37 C 5 CO After 6 hr to overnight replace the transfection medium with 10 ml fresh complete growth medium and incubate at 37 C 5 CO for an additional 24 48 hr Virus titers will generally be highest 48 hr after the start of transfection Caution discarded medium contains infectious retrovirus 9 Harvest the retroviral supernatants and pool similar stocks if desired Caution supernatants contain infectious retrovirus Centrifuge briefly 500 x g for 10 min or filter through a 0 45 um filter to remove cellular debris NOTE The filter used should be made of cellulose acetate or polysulfonate low protein binding instead of nitrocellulose Nitrocellulose binds proteins present in the membrane of retrovirus and destroys the virus 10 Verify virus production by titrating the virus stock see Section VIII then use the virus to transduce target cells or aliquot a
16. with a Fluorescent Protein Reporter RNAi Ready pSIREN RetroQ DsRed Express Cat No 632487 and RNAi Ready pSIREN RetroQ ZsGreen1 Cat No 632455 contain Living Colors fluorescent protein reporters to allow you to directly monitor the delivery efficiency of your gene silencing construct c Tet Inducible Expression of shRNA Using a Retroviral System The Knockout Tet RNAi System H Cat No 630925 and Knockout Tet RNAi System P Cat No 630926 are tetracycline inducible shRNA systems that allow you to tightly regulate the expression of functional short hairpin RNAs shRNAs in mammalian cells in order to silence target genes These systems are especially useful in cases where suppression of a gene may be lethal 7T Retroviral Cell Cycle Reporter Vectors Fucci cell cycle vectors express fluorescent ubiquitination based reporters known as Fucci which allow you to identify cells in various phases of the cell cycle and visualize cell shape Retroviral Fucci vectors include pRetroX G1 Red Cat No 631463 which allows you to identify cells in G1 phase and pRetroX SG2M Cyan Cat No 631462 pRetroX SG2M Red Cat No 631465 and pRetroX SG2Mcyto Red Cat No 631464 which allow you to identify cells transitioning between S G2 and M phases www clontech com Clontech Laboratories Inc A Takara Bio Company Page 6 of 31 Retroviral Gene Transfer and Expression User Manual Constitutive promoter with antibiotic selection Express y
17. 317 Tet System Approved FBS US Sourced Cat No 631105 Retro X Tet Express Inducible Expression System Cat No 631190 1 each 1 each 1 ml 100 rxns 50 ml 25 rxns Retro X Tet Express Vector Set Cat No 631194 not sold separately 20ul pRetroX TRE3G Vector 500 ng ul 20ul pRetroX TRE3G Luc Control Vector 500 ng ul Retro X Universal Packaging Vector Set Cat No 631457 not sold separately 20yl p10A1 Vector 500 ng ul 20yl pAmpho Vector 500 ng ul 20yl pEco Vector 500 ng ul 20ul pVSV G Vector 500 ng ul 20 ul pQCLIN Retroviral Vector 500 ng ul GP2 293 Packaging Cell Line 2 x 10 cells ml Cat No 631458 not sold separately Xfect Transfection Reagent Cat No 631317 Tet System Approved FBS US Sourced Cat No 631105 Tet Express Cat No 631177 C Retroviral Protein Stabilization Destabilization Systems Retro X ProteoTuner Shield System N Cat No 632171 20 ug 60 ul pRetroX PTuner Vector Cat No 632169 not sold separately Shield1 60 ul Cat No 631037 Retro X ProteoTuner Shield System N w ZsGreen1 Cat No 632167 061113 20 ug 60 ul pRetroX PTuner IRES Vector Cat No 631035 not sold separately Shield1 60 ul Cat No 631037 www clontech com Clontech Laboratories Inc A Takara Bio Company Page 11 of 31 Retroviral Gene Transfer and Expression User Manual D Retroviral Vectors amp Systems for RNAi shRNA Gene Knockdown RNAi Read
18. 93 and RetroPack PT67 15 ug retroviral plasmid NOTES e Always add your plasmid s to the Xfect Reaction Buffer before adding Xfect Polymer e Atleast 50 of the solution must consist of Xfect Reaction Buffer Mix well by vortexing for 5 sec at high speed Add the following amounts of Xfect Polymer for the indicated cell lines to the diluted retroviral plasmid DNA and mix well by vortexing for 10 sec at high speed e GP2 293 9 ul Xfect Polymer e AmphoPack 293 EcoPack 2 293 and RetroPack PT67 4 5 ul Xfect Polymer NOTE Always keep the ratio of Xfect Polymer DNA the same Use 0 3 ul of Xfect Polymer per 1 ug of plasmid DNA Incubate DNA Xfect mixture for 10 min at room temperature to allow nanoparticle complexes to form Add the entire 600 ul of DNA Xfect solution Step 6 dropwise to the cell culture medium from Step 1 Rock the plate gently back and forth to mix NOTE It is not necessary to remove serum from your cell culture medium It is normal for the medium to change color slightly upon addition of the DNA Xfect solution Incubate the plate at 37 C 5 CQO After 4 hr to overnight replace the transfection medium with 10 ml fresh complete growth medium and incubate at 37 C for an additional 24 48 hr Virus titers will generally be highest 48 hr after the start of transfection Caution discarded medium contains infectious retrovirus Harvest the retroviral supernatants and pool similar stocks if desired Caution supernata
19. Clontech Laboratories Inc Retroviral Gene Transfer and Expression User Manual Cat Nos Many PT3132 1 061113 Clontech Laboratories Inc A Takara Bio Company 1290 Terra Bella Avenue Mountain View CA 94043 USA U S Technical Support tech clontech com United States Canada Asia Pacific Europe Japan 800 662 2566 1 650 919 7300 33 0 1 3904 6880 81 0 77 543 6116 Retroviral Gene Transfer and Expression User Manual Table of Contents IB Dtod cllonasssisssklaass A 3 HL Listof Components toe cire e n e c e a ona a re ePi a 10 IIl Additional Materials Required eiie eren erant eerta tert ror ege rod p tere E EE La eoa En iskandar 13 IV Safety Guidelines for Working with Retroviruses eese nennen nnne nnne tenente enne enne 15 V Plasmid Vector Manipulations if Ret EE cH d e e a t HE Ene Fre ERI e aere E SPUREN EEE REEE 17 VI Working with Retroviral Packaging Cell Lines a aaaaiaaaaassasaassansansnnsansansnnnannnnnnnnnnnnannnnnnnnnnnnnnnnnnnnsnnnnnnansnnnannsnina 18 A General Cell Culture and Retrovirus Information aaaaaaavassaasassassansaasansaasnnnaannnnnansnnnnnnannnnnnnnnnnnnnnnnnansnninnnnnina 18 B Protocol Reviving Retroviral Packaging Cell Lines from Frozen Stocks esee 19 C Protocol Freezing Retroviral Packaging Cell Line Stocks aaaaaaaaaaasaasaasaasaa
20. amp 631456 to increase your available titer up to 100 fold without ultracentrifugation Concentrated virus allows you to infect target cells at higher MOIs without making more virus or transfecting additional packaging cells E Transduction Enhancers Use Polybrene hexadimethrine bromide Sigma Aldrich No H9268 Lenti X Accelerator see below or RetroNectin see below e Lenti X Accelerator is a magnetic bead based technology designed to accelerate lentiviral and retroviral transduction experiments visit www clontech com for details e RetroNectin is a multivalent molecule that simultaneously binds virus particles and cell surface proteins maximizing cell virus contact RetroNectin in particular is recommended for increasing the transduction efficiency of suspension cells and stem cells visit www clontech com for details Cat No Transduction Enhancer Size 631256 Lenti X Accelerator 400 ul 631257 Lenti X Accelerator 1 000 ul 631254 Lenti X Accelerator Starter Kit each T110A RetroNectin Precoated Dish 10 dishes T100B RetroNectin Recombinant Human Fibronectin Fragment 2 5 mg T100A RetroNectin Recombinant Human Fibronectin Fragment 0 5 mg 061 113 www clontech com Clontech Laboratories Inc A Takara Bio Company Page 14 of 31 Retroviral Gene Transfer and Expression User Manual F Antibiotics for Selecting Transduced Cells Table 3 Recommended Antibiotic Concentrations for Selecting amp Maintaining Stable Cell Lines
21. been reviewed and approved by the Clontech Quality Assurance Department 061 113 www clontech com Clontech Laboratories Inc A Takara Bio Company Page 31 of 31
22. centrated viral dilutions Step 4 to the appropriately labeled wells The final Polybrene concentration will be 4 ug ml in 3 ml Centrifuge the cultures to improve transduction efficiency Culture Centrifugation During Infection Increases Transduction Efficiency Centrifuging the plate at 1 200 x g for 60 90 min at 32 C can significantly increase infection efficiency A room temperature centrifuge is acceptable if a 32 C unit is not available After infecting for 8 24 hours remove the supernatants and begin antibiotic selection using the concentration of antibiotic that is optimal for your cell line Section III F Caution discarded medium contains infectious retrovirus Allow drug resistant colonies to form for 7 14 days Change media every 2 days or add fresh antibiotic every fourth day to maintain selection pressure Stain the colonies with 1 crystal violet solution in 1096 ethanol and count The titer of the virus stock corresponds to the number of colonies generated by the least concentrated dilution multiplied by the dilution factor For example the presence of 4 colonies in the 10 dilution would represent a titer of 4 x 10 colony forming units www clontech com Clontech Laboratories Inc A Takara Bio Company Page 26 of 31 Retroviral Gene Transfer and Expression User Manual IX 061113 Protocol Concentrating Virus Using Retro X Concentrator Retro X Concentrator Section IIL D is a reagent
23. choice of the following Retro X retroviral vectors vector sets and systems Figure 1 1 Constitutive Retroviral Vectors with Antibiotic Selection These retroviral vectors which are derived from the Moloney murine leukemia virus MMLV allow you to express your transgene from a constitutive promoter CMV SV40 or LTR and select for stable integration using antibiotics a Q Series Retroviral Vectors and Vector Sets e Clontech s Q Series of vectors contain a CMV MSV hybrid promoter in the 5 LTR to drive high titers during the packaging step They also contain a self inactivating 3 LTR to reduce promoter interference and drive high expression of your transgene from the internal CMV promoter Expression of an antibiotic resistance gene is achieved through ribosome binding to an internal ribosome entry site IRES e The Retro X Q Vector Set Cat No 631516 gives you a choice of Q Series vector backbones with alternative resistance markers for selection with G418 pQCXIN hygromycin pQCXIH or puromycin pQCXIP b L Series Retroviral Vectors Vector Sets and Systems e pLNCX2 and pLXSN Cat Nos 631503 amp 631509 are basic retroviral vectors that utilize wild type LTRs from MMLV These vectors allow you to express your gene of interest from either a CMV promoter pLNCX2 or the 5 LTR promoter pLXSN Integration of these constructs into the genome of your target cell can be selected for by adding G418 to your culture medium
24. cription from the inducible Prrescv promoter in pRetroX TRE3G which controls expression of your gene of interest cloned downstream of this promoter Tet Inducible Expression Using a Single Retroviral Vector The Retro X Tet Express Inducible Expression System Cat No 631190 is a faster simpler adaptation of the Retro X Tet On 3G Inducible Expression System requiring only a single retroviral vector pRetroX TRE3G with your gene of interest cloned downstream of the inducible Prrescv promoter Transduced cells are induced to express your transgene by adding Tet Express self transducible protein to the culture medium This protein specifically binds to and activates the PrREscv promoter Inducible Protein Stabilization Destabilization ProteoTuner Systems These vectors allow you to fuse your protein to a destabilization domain DD and rapidly stabilize destabilize the resulting fusion protein by adding removing Shield1 from your culture medium a Inducible Protein Stabilization Destabilization Using a Retroviral System The Retro X ProteoTuner Shield System N Cat No 632171 includes the pRetroX PTuner Vector which is designed to express a protein of interest tagged on its N terminus with a mutant FKBP destabilizing domain DD If transfected into mammalian cells it expresses the protein of interest as a fusion protein with the DD Inducible Protein Stabilization Destabilization Using a Retroviral System with a Fluorescent Prote
25. d and expressed by EcoPack 2 293 and recognizes the ecotropic receptor mCAT1 found only on the surface of these cell types NOTE It is possible to transduce human cells with ecotropic retrovirus provided that the cells are pretreated with Ecotropic Receptor Booster Cat No 631471 Section III J Ecotropic Receptor Booster consists of a vial of concentrated exosome like vesicles that are densely coated with the mCAT 1 receptor protein When the booster vesicles are applied to your cells they fuse with the plasma membrane resulting in an increased level of the receptor on the cell surface The AmphoPack 293 Cell Line Cat No 631505 can be used to produce recombinant MMLV based viral particles that infect a broad range of mammalian cells including human mouse and rat cells AmphoPack 293 is also derived from an HEK 293 based cell line that is easily transfected to produce high viral titers The viral envelope protein recognizes the amphotropic receptor Ram 1 The RetroPack PT67 Cell Line Cat No 631510 is the recommended packaging cell line for creating a stable producer cell line for continuous production of retrovirus This retroviral packaging system includes an NIH 3T3 based packaging cell line that expresses the 10A1 viral envelope Virus packaged using RetroPack PT67 cells can be used to infect a broad range of mammalian cells including human mouse and rat cell types The virus is able to enter cells via two different surface mol
26. e The Retro X System Cat No 631508 includes the L Series retroviral expression vectors pLNCX2 and pLXSN Clontech s RetroPack PT67 packaging cell line is also included in this system e The Pantropic Retroviral Expression System Cat No 631512 contains vectors designed for use with the included GP2 293 retroviral packaging cell line and pVSV G envelope vector The system includes the Retro X Pantropic Vector Set which allows you to express your gene of interest from an hsp70 promoter pLNHX or a 5 LTR promoter pLXRN e pLXIN Cat No 631501 is a bicistronic retroviral vector containing an internal ribosome entry site IRES The 5 LTR thus regulates expression of a gene of interest and the neomycin resistance gene on the same bicistronic message 061 113 www clontech com Clontech Laboratories Inc A Takara Bio Company Page 3 of 31 Retroviral Gene Transfer and Expression User Manual 061113 c Vectors Optimized for Expression in Stem Cells The MSCV Murine Stem Cell Virus Retroviral Expression System Cat No 634401 contains vectors that are optimized for introducing and expressing target genes in pluripotent cell lines including murine or human hematopoietic embryonic stem ES and embryonal carcinoma EC cells However they can also be used very effectively with most other mammalian cell lines e These vectors contain a specially designed 5 long terminal repeat LTR from the murine stem cell PCMV virus Th
27. e of Wide range of Target Cells mammalian non Mouse rat cells mammalian cells mammalian cells mammalian cells Table 2 Tropisms associated with Commonly Used Retroviral Envelopes Envelope VSV G 4070A Ampho gap70 Eco 10A1 Dual Tropism Pantropic Amphotropic Ecotropic Dualtropic Receptor target cell Unknown RAM Pit2 mCAT 1 GALV Pit1 RAM1 Pit2 Human Mouse Rat Hamster Possible Target Cat Cell Types Dog Monkey Avian Fish Insect This listing of the most commonly transduced target cell types is not intended to be exclusive It appears likely that a gp96 chaperone client is responsible for binding Bloor et al 2009 061113 www clontech com Clontech Laboratories Inc A Takara Bio Company Page 9 of 31 Retroviral Gene Transfer and Expression User Manual ll List of Components Store cell lines at 196 C and all other components at 20 C A Constitutive Retroviral Vectors amp Expression Systems Retro X Q Vector Set Cat No 631516 e 20 ug pQCXIN Retroviral Vector 500 ng ul e 20 ug pQCXIH Retroviral Vector 500 ng ul e 20 ug pQCXIP Retroviral Vector 500 ng ul e 20 ug pQCLIN Retroviral Vector 500 ng ul e 100 ul 5 pQC Seq PCR Primer 20 uM e 100 ul 3 pQC Seq PCR Primer 20 uM Other Available Q Series Ret
28. ecules the amphotropic retrovirus receptor Ram 1 and the GALV receptor NOTE If you wish to make retrovirus via transient transfection which is the preferred method for most researchers we recommend the Retro X Universal Packaging System or the AmphoPack 293 or EcoPack 2 293 Cell Lines The Pantropic Retroviral Expression System Cat No 631512 also features the GP2 293 packaging cell line which expresses gag and pol proteins The envelope protein is supplied on a separate plasmid pVSV G which is cotransfected with your transfer vector Delivery using a VSV G envelope is not dependent on a cell surface receptor instead it mediates viral entry through lipid binding and plasma membrane fusion Two transfer expression vectors are included with the Pantropic System giving you the choice of using a retroviral 5 LTR or the Drosophila hsp70 promoter to express your gene of interest www clontech com Clontech Laboratories Inc A Takara Bio Company Page 8 of 31 Retroviral Gene Transfer and Expression User Manual Table 1 Summary of Retroviral Packaging Cell Lines Available from Clontech Cell Line GP2 293 AmphoPack 293 EcoPack 2 293 RetroPack PT67 Cat Nos 631530 631512 631505 631507 631510 Cell Line Origin HEK 293 HEK 293 HEK 293 NIH 3T3 Pantropic ecotropic Tropism amphotropic Amphotropic Ecotropic Dualtropic dualtropic VSV G h Envelopes prs i ECO OF 4070A ampho gap70 eco 10A1 we ee l Wide rang
29. ells through passage to a lower density or addition of a mitogen or switch to a lentiviral format such as the Lenti X System which can transduce nondividing or slowly dividing cells Low transduction efficiency See Section D above Low expression of GOI Promoter may be weak or possibly inactivated in target cells Insert a tissue specific promoter for GOI expression Poor target cell viability Check growth parameters MOI too high i e too much virus used Dilute virus stock titrate the virus Polybrene toxicity Reduce or optimize polybrene concentration reduce infection time Infection is toxic to target cells Packaging cell supernatant or medium is toxic to cells Dilute virus stock using target cell culture medium harvest virus from packaging cells using target cell medium Consider using RetroNectin Reagent and the RetroNectin Bound Virus transduction protocol E Establishment of Stable Cell Lines Untransduced cells do not die at the high antibiotic concentration established via The cells have not been recently passaged so they remain well attached to the plate surface even when they are dead titration in Section III F You have achieved 100 transduction efficiency To determine the appropriate antibiotic concentration use cells that have been split within the last 2 3 days There are no surviving cells after transduction followed by selection
30. elope proteins Mature infectious virions then bud from the cell Step 6 Infectious virions are collected in the medium NOTE Although the virions are infectious they lack several critical genes required for their subsequent replication and production in target cells Separating the viral proteins and supplying them in trans adds a strong measure of safety to virus production since several low frequency recombination events would need to occur in order to regenerate a replication competent viral genome Page 22 of 31 Retroviral Gene Transfer and Expression User Manual 061113 B Protocol Packaging Retroviral Vectors Using Xfect Transfection Reagent NOTE This protocol is applicable to all packaging cell types and can be completed in 2 4 days 1 10 11 Approximately 24 hr before transfection seed 4 5 x 10 cells 100 mm plate in 10 ml of growth medium Make sure that the cells are plated evenly Incubate at 37 C 5 CO overnight Continue to incubate the cells until you are ready to add the transfection mixture in Step 7 The cells should be 80 90 confluent at the time of transfection Thoroughly vortex Xfect Polymer In a microcentrifuge tube dilute your retroviral plasmid DNA with Xfect Reaction Buffer to a final volume of 600 ul Use the following amounts of DNA for the indicated cell lines e GP2 293 15 ug retroviral plasmid 15 ug envelope plasmid e g pVSV G e AmphoPack 293 EcoPack2 2
31. eq PCR Primers 20 uM e 100 ul pLXSN Seg PCR Primers 20 uM MSCV Retroviral Expression System Cat No 634401 e 1each MSCV Vector Set Cat No 631461 not sold separately 20 ul pMSVneo Retroviral Vector 500 ng ul 20yl pMSVhyg Retroviral Vector 500 ng ul 20yl pMSVpuro Retroviral Vector 500 ng ul 100ul 5 MSOV Seq PCR Primer 20 uM 100 pl 3 MSOV Seq PCR Primer 20 uM e iml RetroPack PT67 Cell Line 2 x 10 cells ml also sold separately as Cat No 631510 also sold separately as Cat No 631514 ae wa D wa 061 113 www clontech com Clontech Laboratories Inc A Takara Bio Company Page 10 of 31 Retroviral Gene Transfer and Expression User Manual B Tet Inducible Expression Systems Retro X Tet On 3G Inducible Expression System Cat No 631188 1 each 1 each 1 ml 100 rxns 50 ml Retro X Tet On 3G Vector Set Cat No 631192 not sold separately 20ul pRetroX TRE3G Vector 500 ng ul 20ul pRetroX TRE3G Luc Control Vector 500 ng ul 20ul pRetroX Tet3G Vector 500 ng ul Retro X Universal Packaging Vector Set Cat No 631457 not sold separately 20yl p10A1 Vector 500 ng ul 20yl pAmpho Vector 500 ng ul 20yl pEco Vector 500 ng ul 20ul pVSV G Vector 500 ng ul 20ul pQCLIN Retroviral Vector 500 ng ul GP2 293 Packaging Cell Line 2 x 10 cells ml Cat No 631458 not sold separately Xfect Transfection Reagent Cat No 631
32. g initial seeding Cells appear morphologically different Passage of cell culture is too high old cells Thaw purchase new aliquot of packaging cells C Virus Production Poor transfection efficiency as determined by GOI or marker expression in the packaging cell line Cells plated too densely Plate 4 5 x 10 cells 100 mm plate or fewer if the cells divide rapidly Use at 80 9096 confluency See Section VII Transfection is toxic to cells Use the optimized conditions in Section VII Cells harvested or analyzed too soon after transfection Wait 48 hr after transfection for maximal expression of GOI or marker to determine efficiency Low titers 10 cfu ml Poor transfection efficiency See above Section Concentrate the virus using the Retro X Concentrator Section III D to increase your available titer up to 100 fold without ultracentrifugation Poor quality plasmid Use NucleoBond Xtra Section III H to purify your plasmid Virus was harvested too early Harvest virus 48 72 hr after the start of transfection Vector is too large The limit for efficient packaging function is 8 3 kb from the end of the 5 LTR to the end of the 3 LTR Polybrene is missing or at suboptimal concentration Add Polybrene 4 ug ml during transduction or optimize the concentration 2 12 ug ml Virus was exposed to multiple freeze thaw cycles Each cycle reduces titer
33. han collagen may also provide suitable growth substrates e g poly L lysine but only collagen has been tested at Clontech Once recovered the cells may be cultured directly on tissue culture plastic However if adherence is poor we recommend using only collagen coated vessels To prevent osmotic shock and maximize cell survival perform the following 1 Warm 25 ml of complete culture medium in a 37 C water bath See Section II A for medium composition 2 Thaw the vial of cells rapidly in a 37 C water bath with gentle agitation Immediately upon thawing wipe the outside of the vial with 70 ethanol All of the operations from this point on should be carried out in a laminar flow tissue culture hood under strict aseptic conditions Unscrew the top of the vial slowly and using a pipet transfer the contents of the vial to a 15 ml conical centrifuge tube containing 1 ml of prewarmed medium Mix gently 3 Slowly add an additional 4 ml of fresh prewarmed medium to the tube and mix gently 4 Add an additional 5 ml of prewarmed medium to the tube mix gently Centrifuge at 100 x g for 5 min carefully aspirate the supernatant and GENTLY resuspend the cells in complete medium This method removes the cryopreservative and can be beneficial when resuspending in small volumes However be sure to treat the cells gently to prevent damaging fragile cell membranes 5 Mix the cell suspension thoroughly and add to a suitable culture vessel Gen
34. id vectors provided in this kit into a E coli host strain suitable for viral vectors such as Stellar Electrocompetent Cells Cat No 636765 Consult the Vector Information Packet provided with each Retro X vector for further DNA propagation details To purify plasmid DNA for cloning purposes use a suitable NucleoBond or NucleoSpin Kit See www clontech com for available kits and options Using standard cloning techniques insert your coding sequence into the vector s multiple cloning site MCS Consult the Vector Information Packet provided with each Retro X vector for additional cloning details You can also use the In Fusion HD Cloning Plus system Section IILI which allows PCR products to be easily cloned into any linearized vector NOTE Depending on the Retro X vector selected your GOI sequence cDNA or gene fragment may require an ATG initiation codon In such cases addition of a Kozak consensus ribosome binding site Kozak 1987 may improve expression levels but this is generally not required However the fragment or cDNA must not contain a polyadenylation signal The insertion of such sequences between viral LTRs can cause premature cleavage and polyadenylation during transcription of the viral genome This interferes with the production of viable recombinant virions Coffin et al 1997 Perform a transfection grade midi or maxi scale plasmid DNA preparation for each plasmid that will be transfected into the packaging cells
35. ide only general guidelines for Retrovirus use and mammalian cell culture techniques Perform all steps involving cell culture using sterile technique in a Biosafety Level 2 tissue culture hood that has been approved for use with retroviruses For users requiring more information on retroviruses and mammalian cell culture we recommend the following general references e Retroviruses ed by J M Coffin S H Hughes amp H E Varmus 1997 Cold Spring Harbor Laboratory Press NY e Culture of Animal Cells A Manual of Basic Technique and Specialized Applications 6th Edition ed by R I Freshney 2010 Wiley Liss NY e Current Protocols in Molecular Biology ed by F M Ausubel et al 1995 Wiley amp Sons 061 113 www clontech com Clontech Laboratories Inc A Takara Bio Company Page 18 of 31 Retroviral Gene Transfer and Expression User Manual B Protocol Reviving Retroviral Packaging Cell Lines from Frozen Stocks NOTE This protocol can be completed in 1 hour Frozen cells should be cultured immediately upon receipt or as soon as possible thereafter If culturing is significantly delayed after receipt decreased cell viability may result If cell viability from thawed stocks 1s low grow cells for a longer period of time at higher initial density for the first passage For HEK 293 based cell lines we recommend using collagen coated plates or flasks for efficient culturing of frozen stocks Vessels coated with compounds other t
36. in Reporter The Retro X ProteoTuner Shield System N w ZsGreen1 Cat No 632167 includes the pRetroX PTuner IRES vector a bicistronic retroviral expression vector containing ZsGreenl as a reporter to monitor transfection infection efficiency The vector is designed to express a protein of interest tagged with a mutant FKBP destabilizing domain DD simultaneously with ZsGreen1 a human codon optimized variant of the Zoanthus sp green fluorescent protein ZsGreen from the same transcript in transfected mammalian cells www clontech com Clontech Laboratories Inc A Takara Bio Company Page 5 of 31 Retroviral Gene Transfer and Expression User Manual 061113 6 Retroviral Vectors for RNAi shRNA Gene Knockdown These retroviral vectors express shRNA for gene knockdown and are available with a constitutive U6 promoter with or without a fluorescent protein reporter Please see the Knockout RNAi Systems User Manual for more information on these vectors Locate this manual by searching at www clontech com manuals a Constitutive Expression of shRNA Using a Retroviral System RNAi Ready pSIREN RetroQ Vector Cat No 632487 is a retroviral expression vector based on our Q Series of retroviral vectors that is designed to constitutively express an shRNA from the human U6 promoter for use in targeted gene silencing It is ideal for shRNA delivery into hard to transfect cells b Constitutive Expression of shRNA Using a Retroviral System
37. is LTR differs from the MMLV LTR by several point mutations and a deletion that together enhance transcriptional activation and prevent transcriptional suppression in ES and EC cells As a result the PCMV LTR drives high level constitutive expression of the target gene in stem cells or other mammalian cell lines e This system includes the MSCV Vector Set which gives you a choice of three pMSCYV vectors with alternative resistance markers for selection with neomycin hygromycin or puromycin 2 Constitutive Retroviral Vectors with No Selection a Retroviral Vector for Simultaneous Expression of Two Genes of Interest pQCXIX Cat No 631515 lacks a selection marker but contains two multiple cloning sites one on each side of an IRES to allow simultaneous expression of two transgenes b Retroviral Vector for Library Construction pRetro Lib Cat No 635002 contains Sfi1A and Sfi1B restriction sites flanked by a stuffer fragment to enable simple cloning of a full length cDNA library using Clontech s SMART cDNA Library Construction Kit Cat No 634901 3 Constitutive Retroviral Vectors with Fluorescent Selection These retroviral vectors allow you to monitor your transgene by fusing it with a Living Colors fluorescent protein to track its location or express it separately from an IRES to monitor expression a Retroviral Vectors that Provide Monomeric Fusion Tags pRetroQ AcGFP1 N1 C1 Cat Nos 632505 amp 632506 pRetroQ mCherry N1
38. late target cells in their complete growth medium 12 18 hr before transduction Thaw aliquots of your cleared and titrated retroviral stock or use cleared virus stock freshly prepared from packaging cells Section VII B Step 10 or Section VII C Step 9 Mix gently but do not vortex Note that each freeze thaw cycle will decrease titer by 2 4 fold Adjust the volume of medium in the target cell cultures to accommodate the addition of virus and Polybrene Use sufficient Polybrene to obtain the desired final concentration during the transduction step e g 4 ug ml Dilute the retroviral stock with medium to obtain the desired MOL If titer values are unknown use serial dilutions of the virus stock or supernatant such that the total volume of virus represents no more than 1 3 the final volume of culture medium used for transduction Add viral supernatant to the cells and transduce for 8 24 hr Centrifuge the cultures to improve infection efficiency see Section VIILB Step 5 If you are concerned that exposure to either the Polybrene or to the viral supernatant which contains medium conditioned by the packaging cells may adversely affect your target cells limit the transduction to 6 8 hr Remove and discard the virus containing transduction medium and replace it with fresh growth medium Caution discarded medium contains infectious retrovirus Continue to incubate the cells for 24 48 hr to allow your gene product to accumulate in
39. lls are transduced very efficiently Note that the same virus preparation can yield different apparent titers in different cells lines due to host cell factors that can produce very different transduction efficiencies and hence different titer measurements Thus it is important to use the same cell line when comparing titers across experiments Prepare 20 ml of complete medium and add 60 ul of 4 mg ml Polybrene This concentration of Polybrene 12 ug ml will be eventually diluted 3 fold for a final concentration of 4 ug ml during transduction NOTE Polybrene is a polycation that reduces charge repulsion between the virus and the cellular membrane The optimum final concentration of Polybrene may be determined empirically but generally falls within a range of 2 12 ug ml Excessive exposure to Polybrene 224 hr can be toxic to cells Prepare filtered viral supernatant from the transfected Retro X packaging cells Section VID This is your virus stock Prepare six 10 fold serial dilutions of the virus stock as follows a Add 1 35 ml of medium containing Polybrene from Step 2 to each of six sterile and numbered 1 5 ml microfuge tubes b Add 150 ul of the virus stock from Step 3 to Tube 1 Mix gently c Transfer 150 ul from Tube 1 to Tube 2 and mix Continue making serial dilutions by transferring 150 ul from each successive dilution into the next prepared tube Infect the HT 1080 cells by adding 1 ml from each of the five least con
40. nd store at 80 C If smaller volumes are required for transduction Retro X Concentrator Section III D can be used NOTE Titers can drop as much as 2 4 fold with each freeze thaw cycle Creating a RetroPack PT67 Stable Virus Producing Cell Line If your retroviral vector contains a selection marker you may select for stable clones producing high titer virus NOTE This protocol is recommended only for RetroPack PT67 For 293 based packaging cells we recommend virus production via transient transfection Sections B or C 1 Follow the protocols to transfect your transfer vector Section VILB 1 7 or Section VII C 1 6 2 Incubate the plate overnight at 37 C 5 CO 3 Culture cells for 7 days in medium supplemented with the appropriate antibiotic see Section IILF 4 Isolate at least 20 large healthy clones using cloning cylinders Section II A Transfer the clones to individual wells of a culture plate and continue to expand them A Titer the supernatant of each clone e g using the Retro X qRT PCR Titration Kit see Section VIII 6 Choose at least 2 clones exhibiting the highest titer We recommend using gt 5 x 10 IFU ml as a threshold for screening clones www clontech com Clontech Laboratories Inc A Takara Bio Company Page 24 of 31 Retroviral Gene Transfer and Expression User Manual Vill Determining Retroviral Titer 061113 A Introduction To produce consistent transduction results using a known
41. nts contain infectious retrovirus Centrifuge briefly 500 x g for 10 min or filter through a 0 45 um filter to remove cellular debris NOTE The filter used should be made of cellulose acetate or polysulfonate low protein binding instead of nitrocellulose Nitrocellulose binds proteins present in the membrane of retrovirus and destroys the virus Verify virus production by titrating the virus stock see Section VIII then use the virus to transduce target cells or aliquot and store at 80 C If smaller volumes are required for transduction Retro X Concentrator Section III D can be used NOTE Titers can drop as much as 2 4 fold with each freeze thaw cycle www clontech com Clontech Laboratories Inc A Takara Bio Company Page 23 of 31 Retroviral Gene Transfer and Expression User Manual 061113 C Packaging Protocol Using CalPhos Mammalian Transfection Reagent NOTE This protocol is applicable to all packaging cell types and can be completed in 2 4 days 1 Approximately 24 hr before transfection seed 4 5 x 10 cells 100 mm plate in 10 ml of growth medium Make sure that the cells are plated evenly Incubate at 37 C 5 CO overnight The cells should be 80 90 confluent at the time of transfection 2 Ina 15 ml conical centrifuge tube combine your retroviral plasmid DNA with 155 ul 2M Calcium Solution Use the following amounts of DNA for the indicated cell lines e GP2 293 13 ug retroviral plasmid 13
42. o into suspension quickly Remove cell debris and aggregated virus by low speed centrifugation 500 x g for 10 min at 4 C Titrate sample or store at 70 C in single use aliquots EXPECTED RESULTS For VSV G and ecotropic pseudotyped virus this technique can concentrate up to 100 fold for amphotropic and dualtropic virus the expected maximum concentration level is 50 fold www clontech com Clontech Laboratories Inc A Takara Bio Company Page 27 of 31 Retroviral Gene Transfer and Expression User Manual X Transducing Target Cells with Retro X Viruses NOTE This protocol can be completed in 2 3 days The following protocol is a general method for transducing adherent cell lines such as HT 1080 or HeLa using Polybrene Polybrene is a polycation that reduces charge repulsion between the virus and the cellular membrane The optimum final concentration of Polybrene may be determined empirically but generally falls within a range of 2 12 ug ml However excessive exposure to Polybrene 224 hr can be toxic to cells For cells that are difficult to transduce or sensitive to Polybrene Lenti X Accelerator or RetroNectin Reagent can be used to greatly improve speed and transduction efficiency Section IILE The Lenti X Accelerator has the added benefit of providing a much faster 30 min transduction and is equally effective for retroviral and lentiviral transduction For more information visit www clontech com l 2 061113 P
43. or 500 ng ul e 50m Tet System Approved FBS US Sourced Cat No 631105 E Retroviral Cell Cycle Reporter Vectors Retroviral Fucci Cell Cycle Vectors e 10ug pRetroX G1 Red Vector 500 ng ul Cat No 631463 e 10ug pRetroX SG2M Cyan Vector 500 ng ul Cat No 631462 e 10ug pRetroX SG2M Red Vector 500 ng ul Cat No 631465 e 10ug pRetroX SG2Moyto Red Vector 500 ng ul Cat No 631464 F Retroviral Packaging Systems and Cell Lines Retro X Universal Packaging System Cat No 631530 e 1 each Retro X Universal Packaging Vector Set Cat No 631457 not sold separately 20yl p10A1 Vector 500 ng ul 20yl pAmpho Vector 500 ng ul 20yl pEco Vector 500 ng ul 20ul pVSV G Vector 500 ng ul 20ul pQCLIN Retroviral Vector 500 ng ul e iml GP2 293 Packaging Cell Line 2 x 10 cells ml Cat No 631458 not sold separately 061 113 www clontech com Clontech Laboratories Inc A Takara Bio Company Page 12 of 31 Retroviral Gene Transfer and Expression User Manual 061113 Pantropic Retroviral Expression System Cat No 631512 e 1 each Retro X Pantropic Vector Set Cat No 631460 not sold separately 20ul pVSV G Vector 500 ng ul 20yl pLNHX Vector 500 ng ul 20yl pLXRN Vector 500 ng ul 20yl pLLRN Vector 500 ng ul e iml GP2 293 Packaging Cell Line 2 x 10 cells ml Cat No 631458 not sold separately RetroPack PT67 Cell Line Cat No 631510 e im RetroPack PT67 Cell Line
44. our transgene from a constitutive promoter CMV SV40 or LTR and select for stable integration using antibiotics pQCXIN pQCXIH pQCXIP MCS pLNCX2 MCS v pMSCVneo hyg MCS Y Few IRES Neo Hyg Puro wo MCS uro Tv Poos Neo Hyg Puro Constitutive promoter with fluorescent selection Monitor your tri ansgene by fusing it with a fluorescent protein to track its location or express it separately from an IRES to monitor expression pRetroQ AcGFP1 N1 C1 version also available pRetroQ mCherry N1 C1 version also available pRetroX IRES ZsGreen1 BEI eH mes pRetroX IRES DsRedExpress v mes Constitutive promoter with no selection Two available multiple cloning sites separated by an IRES for constitutive coexpression of two separate transgenes pacxix V M S 7 5 5 M Inducible expression Tet On 3G Tightly controlled expression of your transgene using Clontech s Tet On 3G Inducible Expression System pRetroX Tet3G pRetroX TRE3G V MCS V v ines Neo Inducible expression ProteoTuner Fuse your protein to a destabilization domain DD and rapidly stabilize destabilize by adding removing Shield1 from your culture medium pRetroX PTun er o MCS pRetroX PTun Figure 1 Retroviral vectors for many a
45. pplications 061113 www clontech com Clontech Laboratories Inc A Takara Bio Company Page 7 of 31 Retroviral Gene Transfer and Expression User Manual 061113 C Retro X Retroviral Packaging Cell Lines To produce recombinant retrovirus for target cell infection retroviral transfer vectors must be transfected into retroviral packaging cells that provide the gag pol and env genes in trans Various options are available and all retroviral packaging cell lines and systems are compatible with all retroviral transfer vectors sold by Clontech including MMLV and MSCV based retroviruses Table 1 summarizes the retroviral packaging cell lines available from Clontech and Table 2 lists the host range of retroviruses created with the different types of commonly used envelopes The Retro X Universal Packaging System Cat No 631530 is our premium retroviral packaging system featuring the GP2 293 packaging cell line All four commonly used envelopes are supplied on separate vectors that you cotransfect with your transfer vector Envelope choices include VSV G eco ampho and 10A1 to allow you to choose the tropism that is most appropriate for your target cells see Table 2 The EcoPack 2 293 Cell Line Cat No 631507 is an ecotropic HEK 293 based packaging cell line that is also easy to transfect and produces retrovirus with a tropism that limits infection to mouse and rat cells The viral envelope protein is integrate
46. r and Expression User Manual VII 061113 Retrovirus Production using Packaging Cell Lines A General Considerations 1 Optimizing Retroviral Titer To obtain the highest titers from Retro X Systems adhere strictly to the following protocols especially with respect to e Culture size and volume e DNA amounts e DNA quality use transfection grade plasmid DNA Required Materials amp Precautions For optimal results we recommend using e Xfect Transfection Reagent or CalPhos Mammalian Transfection Reagent Section IIIB e 100 mm culture plates e Transfection grade plasmid DNA e g purified using NucleoBond Xtra Maxi Plus see Section V B e 15 ml polystyrene conical centrifuge tube for the CalPhos transfection protocol see Section IIL A IMPORTANT Perform all steps in a sterile tissue culture hood Retrovirus requires the use of a Biosafety Level 2 facility Recombinant retroviruses pseudotyped with VSV G Ampho or 10A1 are capable infecting human cells Know and use appropriate safety precautions see Section IV Protocol Summary A typical protocol involves transient transfection of a retroviral transfer vector that contains a gene of interest into a cell line that expresses some or all of the viral proteins essential for creating a functional retrovirus The retrovirus supernatant is then harvested 48 hr later Figure 3 shows a typical order of events when using the Retro X Universal Packaging System ww
47. roviral Vectors e 20ug pQCXIX Retroviral Vector 500 ng ul Cat No 631515 LRCX Retroviral Vector Set Cat No 631511 e 20ug pLNCX2 Retroviral Vector 500 ng ul also sold separately as Cat No 631503 e 20ug pLHCX Retroviral Vector 500 ng ul e 20ug pLPCX Retroviral Vector 500 ng ul e 100 ul 5 pLNCX Seg PCR Primers 20 uM e 100 ul 3 pLNCX Seq PCR Primers 20 uM Other Available L Series Retroviral Vectors e 20ug pLXIN Retroviral Vector 500 ng ul Cat No 631501 e 20ug pRetro Lib Vector 500 ng ul Cat No 635002 Vectors with Fluorescent Protein Reporters e 20ug pRetroQ AcGFP1 N1 Vector 500 ng ul Cat No 632505 e 20ug pRetroQ AcGFP1 C1 Vector 500 ng ul Cat No 632506 e 20 ug pRetroQ DsRed Monomer N1 Vector 500 ng ul Cat No 632507 e 20 ug pRetroQ DsRed Monomer C1 Vector 500 ng ul Cat No 632508 e 10 ug pRetroQ mCherry N1 Vector 500 ng ul Cat No 632568 e 10ug pRetroQ mCherry C1 Vector 500 ng ul Cat No 632567 e 20ug pRetroX IRES DsredExpress Vector 500 ng ul Cat No 632521 e 20ug pRetroX IRES ZsGreen1 Vector 500 ng ul Cat No 632520 Retro X System Cat No 631508 e iml RetroPack PT67 Cell Line 2 x 10 cells ml also sold separately as Cat No 631510 e 20 ug pLNCX2 Retroviral Vector 500 ng ul also sold separately as Cat No 631503 e 20 ug pLXSN Retroviral Vector 500 ng ul also sold separately as Cat No 631509 e 20ug pLAPSN Retroviral Vector 500 ng ul e 100 ul pLNCX S
48. saassnanansnnnnnsnnnnnnannnnnnannnnnannnnnnnnanina 20 D Packaging Cell Line Maintenance and Passaging aaaaaaaaaasaaasassaasansaasaanansnnnaannnnnannnnnnnnannannannnnnnnnnnnnnnsnnnnnnanina 20 VII Retrovirus Production using Packaging Cell Lines eese nnne nennen rennen eren 21 EEG CES IE CIO 21 B Protocol Packaging Retroviral Vectors Using Xfect Transfection Reagent aaaaaaaaaasaassass ssassaassnsanssnsaanaa 23 C Packaging Protocol Using CalPhos Mammalian Transfection Reagent sese 24 D Creating a RetroPack PT67 Stable Virus Producing Cell Line eene 24 VIT Determining Retroviral iter fe e rre a i E Ince rei t aee Pa DUE ENDE Fe HACER dU PERS 25 Ay Introduction EE 25 B Protocol Determining Viral Titer Using Antibiotic Selection 2 aaaaasaaaaassasaassassansansansannannnnnnnnnannnannansnninnnanina 26 IX Protocol Concentrating Virus Using Retro X Concentrator aaaaaaaasaasaasaasaasaasanssansnnnansnnnnnnnnnnnnnnnnnnnansnannansnnnnnninina 27 X Transducing Target Cells with Retro X Viruses aaaaaaaaaaasaassasaassasaassnanansannannnnnnnnnnnnnnnannnnnannnnnnnnannnnnnnnnnnnansnnnannanianna 28 XI Troubleshooting Guide iR e aaa eee i a i a a E e BL iaaa 29 XI References a EE sos T EEE EE E E EEE 30 Table of Figures Figure 1 Retroviral vectors for many applicatIOns aaiaasaassasaassasaasaassassaa
49. sansaasannannnnnnnnnnnnannnnnannnnnnnnannnnnnnnnnnnansnnnansnninnnani 7 Figure 2 Advanced features of NucleoBond Xtra Maxi and Midi Columns and NucleoBond Finalizer 18 Figure 3 Retrovirus production using the Retro X Universal Packaging System esee 22 Figure 4 Schematic for titering retrovirus supernatants with the Retro X qRT PCR Titration Kit ssessss 25 Table of Tables Table 1 Summary of Retroviral Packaging Cell Lines Available from Clontech esee 9 Table 2 Tropisms associated with Commonly Used Retroviral Envelopes a aaaaaaaaaaasasassaasansansannannnnnnannnannansnnnnnnannnnna 9 Table 3 Recommended Antibiotic Concentrations for Selecting amp Maintaining Stable Cell Lines 15 061 113 www clontech com Clontech Laboratories Inc A Takara Bio Company Page 2 of 31 Retroviral Gene Transfer and Expression User Manual l Introduction A Gene Transfer and Expression Using Recombinant Retroviruses Recombinant retroviral vectors are highly efficient tools for transferring heritable genetic material into the genome of a broad range of dividing cells Lentiviral or adenoviral systems are recommended for viral delivery to nondividing cell types This User Manual supports many Clontech packaging cell lines retroviral vectors and retroviral expression systems B Retro X Retroviral Vectors Clontech offers a
50. se in titrating virus stocks and cryovials for freezing virus stocks e Crystal violet Sigma Aldrich Co Cat No C3886 1 solution prepared in ethanol for staining colonies of transduced cells in the virus titration protocol see Section VIILB e Cloning cylinders PGC Scientific No CORN31666 31668 or 316610 for isolating clones of stable transductants recommended only for RetroPack PT67 see Section VII D www clontech com Clontech Laboratories Inc A Takara Bio Company Page 13 of 31 Retroviral Gene Transfer and Expression User Manual B Transfection Reagents We recommend using either Xfect Transfection Reagent or CalPhos Mammalian Transfection Reagent for transfection of the retroviral transfer vector into Clontech s packaging cell lines Cat No Transfection Reagent 631317 Xfect Transfection Reagent 100 rxns 631318 Xfect Transfection Reagent 300 rxns 631312 CalPhos Mammalian Transfection Reagent 700 rxns C Retrovirus Titration For accurate and consistent transductions we highly recommend titrating your retroviral stocks The Retro X qRT PCR Titration Kit Cat No 631453 provides a fast and simple qRT PCR based titration method The kit determines viral RNA genome content using qRT PCR and SYBR technologies and titrates virus stocks in 4 hr This kit is compatible with all of Clontech s retroviral vectors except the MSCV vectors D Retrovirus Concentration Use Retro X Concentrator Cat Nos 631455
51. tly rock or swirl the dish flask to distribute the cells evenly over the growth surface and place it in a 37 C humidified incubator 5 10 CO as appropriate for 24 hr 6 The next day examine the cells under a microscope If the cells are well attached and confluent they can be passaged for use If the majority of cells are not well attached continue culturing for another 24 hr Complete attachment of newly thawed cultures of HEK 293 based cell lines may require up to 48 hr 7 Once the culture has been started and the cells are growing normally prepare frozen aliquots to provide a renewable source of cells 061 113 www clontech com Clontech Laboratories Inc A Takara Bio Company Page 19 of 31 Retroviral Gene Transfer and Expression User Manual 061113 C Protocol Freezing Retroviral Packaging Cell Line Stocks To ensure a renewable source of packaging cells we recommend expanding and freezing multiple aliquots soon after thawing or at the earliest passages according to the following protocol 1 2 3 4 7 Expand your cells to multiple 10 cm dishes or T75 flasks Trypsinize and pool all of the cells then count the cells using a hemocytometer Centrifuge the cells at 100 x g for 5 min Aspirate the supernatant Resuspend the pellet at a density of at least 1 2 x 10 cells ml in freezing medium Freezing medium can be purchased from Sigma Cat Nos C6164 amp C6039 or use 70 90 FBS 0 20 medium without
52. usion Cloning Kit 638909 In Fusion HD Cloning Plus 10 rxns 638910 In Fusion HD Cloning Plus 50 rxns 638911 In Fusion HD Cloning Plus 100 rxns J Ecotropic Receptor Booster Ecotropic Receptor Booster Cat No 631471 can temporarily increase the efficiency of transduction when using an ecotropic retrovirus or lentivirus and can even be used to transduce human cells with these viruses 061 113 www clontech com Clontech Laboratories Inc A Takara Bio Company Page 15 of 31 Retroviral Gene Transfer and Expression User Manual IV 061113 Safety Guidelines for Working with Retroviruses The protocols in this User Manual require the production handling and storage of infectious retrovirus It is imperative to fully understand the potential hazards of and necessary precautions for the laboratory Use of retroviruses The National Institute of Health and Center for Disease Control have designated retroviruses such as Moloney murine leukemia virus MMLV as Level 2 organisms This requires the maintenance of a Biosafety Level 2 facility for work involving this virus and others like it MMLV does not naturally infect human cells however virus packaged from the MMLV based vectors described here is capable of infecting human cells The viral supernatants produced by these retroviral systems could depending on your retroviral insert contain potentially hazardous recombinant virus Similar vectors have been approved for human gene
53. w clontech com Clontech Laboratories Inc A Takara Bio Company Page 21 of 31 Retroviral Gene Transfer and Expression User Manual Envelope vector _ 1 Cotransfect your transfer vector e g pVSV G with your chosen envelope vector O into GP2 293 cell line Transfection Xfect or CalPhos 2 Transcription and translation Retro X Transient Transfer Vector expression containing a Viral d ag your transgene T 7 a proteins M 9 eu R a E X GP2 293 3 Viral proteins y coi TM Pol k Cell Line recognize V RNA 4 Assembly of virus cores Env protein 5 Budding of infectious O virions 0 Harvest retrovirus in culture supernatant Figure 3 Retrovirus production using the Retro X Universal Packaging System 061113 www clontech com Clontech Laboratories Inc A Takara Bio Company Step 1 Cotransfect GP2 293 cells with the Retro X vector containing your gene of interest GOI and an envelope plasmid such as pVSV G Step 2 Resulting production of the corresponding recombinant retroviral genome and viral packaging proteins GP2 293 Cells express gag and pol from genomic locations Step 3 Recognition of the packaging sequence VJ on the recombinant viral RNA genome by the packaging proteins Step 4 Resulting assembly of viral cores which are transported to the cell membrane Step 5 Cores are then enveloped by cellular membrane containing aggregated VSV G or other env
54. y pSIREN RetroQ Vector Cat No 631526 20 rxns e 1g RNAi Ready pSIREN RetroQ Vector linearized 25 ng ul e 20ul Luciferase shRNA Annealed Oligonucleotide 0 5 pmol ul e 20ul Negative Control shRNA Annealed Oligonucleotide 0 5 pmol ul RNAi Ready pSIREN RetroQ DsRed Express Vector Cat No 632487 20 rxns e 1g RNAi Ready pSIREN RetroQ DsRed Express Vector linearized 25 ng ul e 20ul Luciferase shRNA Annealed Oligonucleotide 0 5 pmol l e 20ul Negative Control shRNA Annealed Oligonucleotide 0 5 pmol l RNAi Ready pSIREN RetroQ ZsGreen1 Vector Cat No 632455 20 rxns e 1ug RNAi Ready pSIREN RetroQ ZsGreen1 Vector linearized 25 ng ul e 20ul Luciferase shRNA Annealed Oligonucleotide 0 5 pmol l e 20ul Negative Control shRNA Annealed Oligonucleotide 0 5 pmol l Knockout Tet RNAi System H Cat No 630925 e 1 each Tet RNAi Vector Set H Cat No 631125 not sold separately 2ug RNAi Ready pSIREN RetroQ TetH Vector linearized 50 ng ul 20pg ptTS Neo Vector 500 ng ul 20ug pQC tTS IN Vector 500 ng ul 20ug pSIREN RetroQ TetH Luc Vector 500 ng ul e 50ml Tet System Approved FBS US Sourced Cat No 631105 Knockout Tet RNAi System P Cat No 630926 e 1 each Tet RNAi Vector Set P Cat No 631130 not sold separately 2ug RNAi Ready pSIREN RetroQ TetP Vector linearized 50 ng ul 20ug_ ptIS Neo Vector 500 ng ul 20ug pQC tTS IN Vector 500 ng ul 20ug pSIREN RetroQ TetP Luc Vect

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