Lenti-X™ Lentiviral Expression User Manual
Contents
1. Notice to Purchaser cont d Metridia Luciferase Markova S V Golz S Frank L A Kalthof B amp Vysotski E S 2004 Cloning and expression of cDNA for a luciferase from the marine copepod Metridia longa A novel secreted bioluminescent reporter enzyme J Biol Chem 279 5 3212 3117 ProteoTuner Protein Stabilization Destabilization Products Reef Coral Fluorescent Proteins DsRed Express and DsRed Express2 The RCFPs including DsRed Express and DsRed Express2 are covered by one or more of the following U S Patent Nos 7 166 444 7 157 565 7 217 789 7 338 784 7 338 783 7 537 915 6 969 597 7 150 979 and 7 442 522 Retroviral Packaging Systems Use of this product is covered by U S Patent No 5 858 740 and is limited to use solely for research purposes Please contact GBP IP LLC for a license to use these products for commercial purposes RNAi Products This product is covered by US patent No 6 506 559 Tet Based Expression Products Use of the Tetracycline controllable expression systems the Tet Technology is covered by a series of patents including U S Patent Nos 5 464 758 and 5 814 618 which are proprietary to TET Systems GmbH amp Co KG Academic research institutions are granted an automatic license with the purchase of this product to use the Tet Technology only for internal academic research purposes which license specifically excludes the right to sell or otherwise transfer the Tet Tech2. Poor transfection efficiency Concentrate the virus see Appendix A for large vec tors or reduce size of the insert Lenti X Lentiviral Expression Systems User Manual IX Troubleshooting Guide continued Table I Troubleshooting Guide for Lenti X Expression Systems continued D Transduction of Target Cells Transduction protocol not See Appendix B for references to help with optimized optimizing transduction protocols 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 Low viability of target cells RetroNectin Reagent and the RetroNectin Bound during transduction Virus transduction protocol or purify your virus prior to transduction using the Lenti X Maxi Purifi cation Kit Cat Nos 631233 amp 631234 Excessive exposure to polybrene optimize amount of polybrene titrate or shorten exposure time to viral supernatant 1 Poor transduction efficiency Use RetroNectin Reagent or RetroNectin coated plates in the RetroNectin Bound Virus transduction protocol which allows virions to bind the RetroNec tin substratum and be washed free of inhibitors prior to target cell infection or purify your virus prior to transduction using the Lenti X Maxi Purifi cation Kit Cat Nos 631233 amp 631234 Low transduction efficiency See Section D 1 Promoter
3. Xira Midi NuctooBond Xtra Maxi Low silica W resin bed High flow rate Figure 4 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 www clontech com Clontech Laboratories Inc ATakara Bio Company Lenti X Lentiviral Expression Systems User Manual V Cell Culture Guidelines A General Cell Culture and Lentivirus Information The protocols in this User Manual provide only general guidelines for lentivirus use and mammalian cell culture technigues Perform all steps involving cell culture using sterile technigue in a Biosafety Level 2 tissue culture hood that has been approved for use with lentiviruses For users reguiring more information on lentiviruses 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 Culture of Animal Cells 5th Edition ed by R I Freshney 2005 Wiley Liss N
4. User Manual Lenti X Lentiviral Expression Systems User Manual O Clontech United States Canada 800 662 2566 Asia Pacific 1 650 919 7300 Europe 33 0 1 3904 6880 Japan 81 0 77 543 6116 PT5135 1 PROY3745 Clontech Laboratories Inc ATakara Bio Company Published November 2010 1290 Terra Bella Ave Mountain View CA 94043 Technical Support US E mail tech clontech com www clontech com Lenti X Lentiviral Expression Systems User Manual Table of Contents kL introductio Mesnine ee a iN ea aia ea DU RY y DYN YNN Un VEEE 3 A Gene Transfer and Expression Using Recombinant Lentiviruses sssessssessseserserereereresrerrererrrrerrrreens 3 Bo Lenti X Veto rs eis ei aoise CT GYNYG A CH TO GYF FD TF 3 C Lenti X HTX Packaging Systems cseseessesssescsseeeecseseseseeaescseseaescssscececaesececersesenesavscateaeessenteesesaraes 3 Il Additional Materials Required cececceeeeeeeeeeeee cece eeeeeeeeeeaeeaeeeeeeeeeeeeeseeeeeseaaeeeseeeeeeeneees 6 Ill Safety Guidelines for Working with Lentiviruses cccccccseeeeesseeeeeeeseeeeeeeesseeeeeeeneeeees 9 IV Plasmid Vector Manipulations cecccecseeeeeeceeeeeeeeeeeeeeeaeaeeeeeeeeeeeeeeseeeseaaeaeaeeeeseeeeeneees 10 A General Molecular Biology Techniques ccessessssesesssseseseseeseecsesesececsssescsseasscseesesecasaceesasateeeasavaees 10 B Plasmid Vector Propagation amp Construction of Your Customized Lenti
5. Clontechniques XXII 4 1 2 Improve Viral Transductions with RetroNectin Reagent October 2008 Clontechniques XXIII 4 7 8 Inducible Lentiviral Gene Expression Systems October 2007 Clontechniques XXII 4 3 5 Kozak M 1987 At least six nucleotides preceding the AUG initiator codon enhance translation in mammalian cells J Mol Biol 196 947 50 Kwon Y J Hung G Anderson W E Peng C A amp Yu H 2003 Determination of infectious retrovirus concentration from colony forming assay with quantita tive analysis J Virol 77 5712 5720 Lentiviral Expression System October 2007 Clontechniques XXII 4 6 Lentiviral Vectors for CDNA and shRNA Delivery October 2008 Clontechniques XXII 4 1 2 Miyao Y Shimizu K Tamura M Yamada M Tamura K Nakahira K Kuriyama S Hayakawa T amp Ikenaka K 1995 A simplified general method for determination of recombinant retrovirus titers Cell Struct Funct 20 177 183 Morgan R A Cornetta K amp Anderson W F 1990 Applications of the polymerase chain reaction in retroviral mediated gene transfer and the analysis of gene marked human TIL cells Hum Gene Ther 1 135 149 Murdoch B Pereira D S Wu X Dick J E amp Ellis J 1997 A rapid screening procedure for the identification of high titer retrovirus packaging clones Gene Ther 4 744 749 Nelson D M Wahlfors J J Chen L Onodera M amp Morgan R A 1998 Characterization of d
6. PROY3745 16 www clontech com Clontech Laboratories Inc ATakara Bio Company Lenti X Lentiviral Expression Systems User Manual IX Troubleshooting Guide Clontech Laboratories Inc ATakara Bio Company Table I Troubleshooting Guide for Lenti X Expression Systems A Vector Cloning Some viral vectors may undergo rearrangement Use Stellar Electrocompetent Cells Cat No 1 Plasmid is difficult to grow between the 5 and 3 LTRs 636765 or Supercharge EZ10 Electrocompetent Cells or clone when propagated in less Cat No 636756 to produce high DNA yields and to than optimal E coli host minimize the potential for DNA rearrangements strains B Lenti X 293T Packaging Cells Use thawing procedure in Section V B and or consult the Lenti X 293T Cell Line Protocol at a Glance PT4058 2 1 Poor viability upon thawing i Use DMEM with additives listed in Section II B Use Incorrect culture mediuin 10 Tet System Approved FBS Tc free Improper tissue culture Use collagen l coated plates to aid cell adherence plasticware during initial seeding 2 Slow growth incorrectc lture medium Use DMEM with additives listed in Section II B Use i 8 1096 Tet System Approved FBS Tc free 3 Cells do not attach to plate Improper tissue culture Use collagen l coated plates to aid cell adherence plasticware during initial seeding 4 Cells appear morphologi Passage of cell culture is too T cally different high old cells Thaw p
7. PROY3745 9 Lenti X Lentiviral Expression Systems User Manual IV Plasmid Vector Manipulations Protocol No PT5135 1 Version No 10 PROY3745 A General Molecular Biology Techniques These protocols contain only general information for propagating plasmid vectors and for preparing your customized expression construct in a Lenti 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 ed by J Sambrook et al 2001 Cold Spring Harbor Laboratory Press NY B Plasmid Vector Propagation amp Construction of Your Customized Lenti X Vector 1 To ensure that you have a renewable source of plasmid DNA transform each of the plasmid vectors provided in this kit into a coli host strain suitable for viral vectors such as Supercharge EZ10 Electrocompetent Cells Cat No 636756 or Stellar Electrocompetent Cells Cat No 636765 Consult the Vector Infor mation Packet provided with each Lenti X vector for further DNA propagation details 2 To purify plasmid DNA for cloning purposes use a suitable NucleoBond or NucleoSpin Kit See www clontech com for available kits and options 3 Using standard cloning techniques insert your coding sequence into the vector s mul
8. 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 cPPT Element This product and its use are the subject of U S Pat No 6 682 907 The purchase of this product con veys to the buyer the non transferable right to use the purchased amount of the product and components of the product in research conducted by the buyer whether the buyer is an academic or for profit entity The buyer cannot disclose information sell or otherwise transfer this product its components or materials made using this product or its components to a third party or otherwise use this product or its components or materials made using this product or its components for any commercial purposes If the buyer is not willing to accept the limitations of this limited use statement Clontech is willing to accept return of the product with a full refund For information on purchasing a license to the DNA Flap technology for purposes other than research contact the Transfer of Technology Office Institut Pasteur 28 rue du Docteur Roux 75724 Paris France Cedex
9. J M Landazuri N Yarmush M L amp Morgan J R 2001 Complexation of retrovirus with cationic and anionic polymers increases the efficiency of gene transfer Hum Gene Ther 12 13 1611 1621 Morling F J amp Russell S J 1995 Enhanced transduction efficiency of retroviral vectors coprecipitated with calcium phosphate Gene Ther 2 7 504 508 Hennemann B Chuo J Y Schley P D Lambie K Humphries R K amp Eaves C J 2000 High efficiency retroviral transduction of mammalian cells on positively charged surfaces Hum Gene Ther 11 1 43 51 E Flow through transduction concentrating cells and virus together in small culture systems Pan D Shankar R Stroncek D E amp Whitley C B 1999 Combined ultrafiltration transduction in a hollow fiber bioreactor facilitates retrovirus mediated gene transfer into peripheral blood lymphocytes from patients with mucopolysaccharidosis type II Hum Gene Ther 10 17 2799 28 10 Chuck A S amp Palsson B O 1996 Consistent and high rates of gene transfer can be obtained using flow through transduction over a wide range of retroviral titers Hum Gene Ther 7 G 743 750 F Addition of fibronectin adhesion domains within fibronectin allow binding to both target cells and virions to facilitate colocalization Zhou P Lee J Moore P amp Brasky K M 2001 High efficiency gene transfer into rhesus macaque primary T lymphocytes by combining 32 degre
10. Proc Natl Acad Sci USA 92 17 7739 7743 Zhou P Lee J Moore P Brasky K M 2001 High efficiency gene transfer into rhesus macaque primary T lym phocytes by combining 32 degrees C centrifugation and CH 296 coated plates effect of gene transfer protocol on T cell homing receptor expression Hum Gene Ther 12 15 1843 1855 Kotani H Newton P B 3rd Zhang S Chiang Y L Otto E Weaver L Blaese R M Anderson W F amp McGarrity G J 1994 Improved methods of retroviral vector transduction and production for gene therapy Hum Gene Ther 5 1 19 28 Higashikawa F amp Chang L 2001 Kinetic analyses of stability of simple and complex retroviral vectors Virology 280 1 124 131 B Centrifugation during transduction spinoculation may counteract diffusion of virus when binding target cells Bunnell B A Muul L M Donahue R E Blaese R M amp Morgan R A 1995 High efficiency retroviral mediated gene transfer into human and nonhuman primate peripheral blood lymphocytes Proc Natl Acad Sci USA 92 17 7739 7743 Ohkubo T Barcena A Smith C A Harrison M R amp Muench M O 2001 High efficiency retroviral trans duction of fetal liver CD38 CD34 cells implications for in utero and ex utero gene therapy Fetal Diagn Ther 16 5 299 307 Movassagh M Boyer O Burland M C Leclercq V Klatzmann D amp Lemoine F M 2000 Retrovirus me dia
11. V Petit C Guetard D Nerhbass U Montagnier L amp Charneau P 2000 HIV 1 genome nuclear import is mediated by a central DNA flap Cell 101 173 185 Zufferey R Donello Trono D amp Hope T J 1999 Woodchuck hepatitis virus posttranscriptional regulatory element enhances expression of transgenes delivered by retroviral vectors J Virol 73 2886 2892 Clontech Laboratories Inc www clontech com Protocol No PT5135 1 ATakara Bio Company Version No PROY3745 19 Lenti X Lentiviral Expression Systems User Manual Appendix A Additional Protocols Protocol No PT5135 1 Version No 20 PROY3745 A Protocol Titrating Antibiotics for Selecting Stable Cell Lines Prior to using the antibiotics G418 Cat No 631308 and or puromycin Cat No 631306 to select cells that have been either singly or doubly transduced with Lenti X lentiviruses it is necessary to titrate each selection agent to determine the optimal concentration for your target cell line With HeLa cells for example we have found 400 pg ml G418 and 1 0 pg ml puromycin to be optimal Table ll Recommended Concentrations for Selection Antibiotics ug ml Puromycin 0 25 2 0 5 10 e For selecting stable transformants with G418 and hygromycin B use the lowest concentration that results in massive cell death in 5 days and kills all the cells within two weeks e Puromycin selection occurs more rapidly use a concentration that will kill a
12. the viral genome during target cell infection The cPPT CTS element improves vector integration and transduction efficiency Zennou ef 4 2000 e RRE A Rev response element helps to increase titers by promoting the nuclear export of unspliced viral genomic RNA Cochrane ef al 1990 C Lenti X HTX Packaging Systems To produce recombinant lentivirus for target cell infection Lenti X plasmid vectors must be cotransfected into Lenti X 293T cells along with a Lenti X HTX Packaging Mix in order to assemble your vector and accompanying GOI into infectious virions Figure 2 Highest Titers Lenti X HTX Packaging Mixes are plasmid mixtures that provide the necessary viral packag ing components in specific optimized ratios When your vector and packaging mix are transfected into Lenti X 293T cells using the Xfect transfection reagents the packaging mix expresses the Pol RT amp IN Tat Rev and Gag lentiviral proteins and either the VSV G envelope protein or the ecotropic gp70 envelope protein from MLV Wu et al 2000 Clontechniques October 2007 The recombinant viral vector is then replicated and assembled into complete pseudotyped virus particles Figure 2 The packaging mix includes an expression vector for the Tet Off transcriptional activator tTA and uses Tet transactivation to produce very high expression levels of specific viral proteins Gossen amp Bujard 1992 This optimized expression strat egy combined with h
13. 15 www pasteur fr DsRed Monomer and Fruit Fluorescent Proteins The DsRed Monomer and the Fruit Fluorescent Proteins are covered by one or more of the following U S Patents 7 005 511 7 157 566 7 393 923 and 7 250 298 GoStix Products Lentiviral Expression Products Portions of this product are covered by several patent applications owned by or licensed to Open Biosystems Inc The purchase of this product conveys to the buyer the limited non exclusive non transferable right without the right to resell repackage or further sublicense under these patent rights to perform the viral infection methods using the lentiviral vectors claimed in those patent applications for research purposes solely in conjunction with this product No other license is granted to the buyer whether expressly by implication by estoppel or otherwise In particular the purchase of this product does not include nor carry any right or license to use develop or otherwise exploit this product commercially and no other rights are conveyed to the buyer to use the product or components of the product of any other purposes including without limitation provision of services to a third party generation of commercial databases or clinical diagnostics or therapeutics This product is sold pursuant to a license from Open Biosystems Inc and Open Biosystems Inc reserves all other rights under these patent rights For information on purchasing a license to the patent ri
14. 95 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 od ohs biosfty bmbl5 bmbl5toc htm Biosafety Level 2 The following information is a brief description of Biosafety Level 2 Jf 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 lentiviruses Summary of Biosafety Level 2 e 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 defining any needed waste decontamination or medical surveillance policies 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 unrecir culated PPE protective laboratory coats gloves face protection as needed e Facilities Autoclave available for waste decontamination Chemical disinfectants available for spills www clontech com Protocol No PT5135 1 Version No
15. Additional Materials Reguired A Cell Lines for Lentivirus Packaging and Titration Lenti X 293T Cell Line Cat No 632180 This is an HEK 293T derived cell line optimized for Lenti X virus production Io obtain high titer supernatants of infectious lentivirus your Lenti X vector and a Lenti X HTX Packaging Mix are cotransfected into Lenti X 293T cells using the Xfect transfection reagent The transfected cells will consistently produce very high titers of pseudotyped lentivirus HT 1080 cell line American Type Culture Collection HT 1080 ATCC No CCL 121 Recommended This cell line is easily transduced by recombinant lentiviruses and is frequently used for lentiviral titration Alternatively virus stocks can be titrated with the Lenti X qRT PCR Titration Kit Cat No 632165 or the Lenti X p24 Rapid Titer Kit Cat No 632200 Or you can save time and test the quality of your lentiviral supernatant in 30 seconds using Lenti X Go Stix Cat Nos 631241 631243 amp 631244 B Mammalian Cell Culture Supplies Lenti X 293T Cell Line growth medium 90 Dulbecco s Modified Eagle s Medium DMEM with high glucose 4 5 g L 4 mM L glutamine and 3 7 g L sodium bicarbonate Sigma Aldrich Co No D5796 and 10 tetracycline free fetal bovine serum Add 1 mM sodium pyruvate HT 1080 growth medium 90 Dulbecco s Modified Eagle s Medium DMEM with high glucose 4 5 g L 4 mM L glutamine and 3 7 g L sodium bicarbonate Si
16. C Remove the supernatant 3 Resuspend the virus to 0 5 190 of the original volume in TNE 50 mM Tris HCl pH 7 8 130 mM NaCl 1 mM EDTA and incubate overnight at 4 C Note If desired perform a second round of ultracentrifugation Steps 1 2 4 Determine the viral titers of pre and post concentrated viral supernatants 5 Iransduce target cells www clontech com Clontech Laboratories Inc ATakara Bio Company Lenti X Lentiviral Expression Systems User Manual Appendix B Additional Viral Infection Methods Clontech Laboratories Inc ATakara Bio Company These references are provided for fine tuning your transduction protocols so that you may improve your transduction efficiency in target cells This list is not a comprehensive list but many of these protocols will work for a wide range of cell types You must determine which methods work best for your targets and certain methods may have additive effects For optimization experiments we recommend using one of our Lenti X Fluorescent Vectors to express a Living Colors Fluorescent Protein which simplifies the detection and quantitation of lentiviral gene transfer efficiency A Transduction of cells at 32 C Decreasing temperature increases viral half life during trans duction Bunnell B A Muul L M Donahue R E Blaese R M Morgan R A 1995 High efficiency retroviral me diated gene transfer into human and nonhuman primate peripheral blood lymphocytes
17. Clontech These sera have been functionally tested in our Tet Systems and found to be free of contaminating Tc activity 15 x 10 4 10 x 10 5 Fold induction 5x 10 Tet System Other commercially Approved FBS available FBS Figure 3 Tetracycline activity in bovine sera The CHO AA8 Luc Tet Off Control Cell Line was grown in media prepared with different lots of FBS Average uninduced expression level 0 21 RLU n 21 S D 0 07 maximum expression levels varied from 123 to 3 176 RLU www clontech com Clontech Laboratories Inc ATakara Bio Company Lenti X Lentiviral Expression Systems User Manual ll Additional Materials Required continued Clontech Laboratories Inc ATakara Bio Company B Mammalian Cell Culture Supplies continued e Sodium pyruvate solution 100 mM sterile filtered Sigma Aldrich Co No 8636 for supplementing cell culture media e Penicillin streptomycin solution of 10 000 units ml penicillin G sodium and 10 000 pg ml streptomycin sulfate 100X Sigma Aldrich Co No P0781 Trypsin EDTA Trypsin Sigma Aldrich Co No T3924 e Dulbecco s phosphate buffered saline DPBS Sigma Aldrich Co No D8662 e L glutamine solution 200 mM sterile filtered Sigma Aldrich Co No G7513 Optional e Cell Freezing Medium with or without DMSO Sigma Aldrich Co No C6164 or No C6039 e Tissue culture plates 100 mm for packaging cell transfections other plates and flasks as require
18. RINS technique to titer recombinant virus and evaluation of the efficiency of viral transduction Anal Biochem 291 96 101 Cochrane A W Chen C H amp Rosen C A 1990 Specific interaction of the human immunodeficiency virus Rev protein with a structured region in the env mRNA Proc Natl Acad Sci U S A 87 1198 202 Coffin J M Hughes S H amp Varmus H E eds 1997 Retroviruses Cold Spring Harbor Laboratory Press Cold Spring Harbor NY Fluorescent Lentiviral Expression Vectors 2007 Clontechniques XXII 4 7 Freshney R I 2005 Culture of Animal Cells 5th Edition Wiley Liss New York NY Goff S Traktman P amp Baltimore D 1981 Isolation and properties of Moloney murine leukemia virus mutants use of a rapid assay for release of virion reverse transcriptase J Virol 38 239 248 Gossen M amp Bujard H 1992 Tight control of gene expression in mammalian cells by tetracycline responsive promoters Proc Natl Acad Sci USA 89 5547 5551 Higashikawa F amp Chang L 2001 Kinetic Analysis of stability of simple and complex retroviral vectors Virology 280 124 131 Higashimoto T Urbinati F Perumbeti A Jiang G Zarzuela A Chang L J Kohn D B amp Malik P 2007 The woodchuck hepatitis virus post transcription al regulatory element reduces readthrough transcription from retroviral vectors Gene Ther 14 17 1298 1304 High Efficiency Lentiviral Packaging October 2007
19. X Vector 10 V Cell Culture Guidelines cccccccceceeeeeeecesececeeeesaeananssssseeseeeeeeeeaeeeeeeeseususcesesesensesasananesss 11 A General Cell Culture and Lentivirus Information ueueuuu eu AIL AIII II LIII I 11 B Protocol Starting Lenti X 293T Cell Line Cultures from Frozen Stock s ssssessssessssesssesesreserrereresrerese 11 VI Producing Lentivirus from Lenti X Vectors ccccceeeeeeeeeeeeeeseeeeeeeeseeneeeeeesseeeeeeeeeseeaeeees 12 A Protocol Using the Lenti X HTX Packaging System to Produce Lentiviral Supernatants 12 VII Determining Lentiviral Titer unuuuueuu iii iiL LLssssy LA sneer eee eeaseaaeeeseeeeeeeeeseeseeeseaaeeeseeees 13 A dutrod uc toi Y Y A Y I WY Y SR 13 B Protocol Determining Viral Titer Using Antibiotic Selection wc ceccseeeesseeesseseseseeseseecseeeeesesataeeees 14 C Alternative Titration Methods cccccccccccessccssseesseesseccseceesecessseseecsssecssecessesssscessceseeceasseseecesseesaeessees 15 VIII Transducing Target Cells with Lenti X Viruses cccccceceeceeeeeeseeeeeeeeeeeeeeeeseeeeeeenaaeeeeeeees 16 A Protocol Transducing Target Cells with Lenti X Viruses ccceecsseeseesssesesseseseseeseececseeceeserseseeesenaees 16 IX Troubleshooting Guide icciiccscescecc ccs secsicccesssceveccaseicevecessccvevecestcteveceesttevvecsssstevecensestevsensene 17 Ms ReferenCB Sr eii ini Gu YL hcheassadnewiavasseazasve
20. Y Current Protocols in Molecular Biology ed by F M Ausubel et al 1995 Wiley amp Sons B Protocol Starting Lenti X 293T Cell Line Cultures from Frozen Stock 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 For HEK 293 based cell lines we recommend using collagen a a coated plates or flasks for efficient culturing of frozen stocks Vessels coated with compounds other than 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 B for medium composition Note Be sure to use Tet System Approved Fetal Bovine Serum Cat Nos 631101 amp 631106 when using these cells with the Lenti X HTX Packaging System Cat Nos 631247 amp 631249 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 t
21. c www clontech com Protocol No PT5135 1 ATakara Bio Company Version No PROY3745 11 Lenti X Lentiviral Expression Systems User Manual VI Producing Lentivirus from Lenti X Vectors Protocol 2 4 days E o Attention STOP Don t forget Tet System Approved FBS 100 mm culture plates Transfection grade DNA These 293T cells were plated at an optimal transfection density An example of 293T cells at virus harvest shown here using a transfer vector containing ZsGreen1 Protocol No PT5135 1 Version No PROY3745 12 A Protocol Using the Lenti X HTX Packaging System to Produce Lentiviral Supernatants To obtain the highest titers from the Lenti X HTX Packaging System use the Lenti X 293 T Cell Line and adhere strictly to the following protocol especially with respect to 1 culture size and volume 2 DNA amounts and transfection grade quality 3 tetracycline free serum in Lenti X 293T growth media and 4 incubation times All Xfect transfection reagents volumes and conditions are optimized for use with Lenti X Vectors the Lenti X HTX Packaging Mix and Lenti X 293T cells Use 100 mm tissue culture plates and be sure to use Tet System Ap proved FBS guaranteed Tc free both in the transfection medium Step 1 and in the medium used to collect the virus Step 8 Tetracycline contaminated serum is detrimental to the expression of essential packaging components in the Le
22. d e Polystyrene culture tubes 12 x 75 mm e g BD Falcon No 352054 for packaging cell transfections Sterile microfuge tubes 1 5 ml for use in titrating virus stocks and cryovials for freezing virus stocks e Crystal violet Sigma Aldrich Co No C3886 1 solution prepared in ethanol for staining colonies of transduced cells in the virus titration protocol Section VII B e Cloning cylinders PGC Scientific No CORN31666 31668 or 316610 for isolating clones of stable transductants C Lentivirus Titration Kits For accurate and consistent transductions we highly recommend titrating your lentiviral stocks The Lenti X qRT PCR Titration Kit Cat No 631235 provides a fast and simple gRI PCR based titration method Clontechniques January 2008 The kit determines viral RNA genome content using qRT PCR and SYBR technologies and titrates virus stocks in 4 hr The Lenti X p24 Rapid Titer Kit Cat No 632200 uses ELISA to specifically measure the amount of p24 capsid protein present in your viral supernatant and then correlates the level of p24 directly to virus titer Alternatively you can use Lenti X GoStix Cat Nos 631241 631243 amp 631244 to test the quality of your viral supernatant in just 30 seconds The GoStix detect lentiviral p24 in only 20 pl and can be used to determine whether virus production is within a usable range or for selecting the best time to harvest your virus D Lentivirus Purificat
23. d 84 3 780 788 H Use of cationic liposomes Enhance virus to cell fusion Kaneko Y amp Tsukamoto A 1996 Cationic liposomes enhance retrovirus mediated multinucleated cell formation and retroviral transduction Cancer Lett 105 1 39 44 Porter C D Lukacs K V Box G Takeuchi Y amp Collins M K 1998 Cationic liposomes enhance the rate of transduction by a recombinant retroviral vector in vitro and in vivo J Virol 72 6 4832 4840 www clontech com Clontech Laboratories Inc ATakara Bio Company Lenti X Lentiviral Expression Systems User Manual Appendix B Additional Viral Infection Methods continued Clontech Laboratories Inc ATakara Bio Company I Use of histone deacetylase inhibitors to increase titer Relieves repression of viral expres sion by hyperacetylation of histones Chen W Y Bailey E C McCune S L Dong J Y amp Townes T M 1997 Reactivation of silenced virally trans duced genes by inhibitors of histone deacetylase Proc Natl Acad Sci USA 94 11 5798 5803 Tobias C A Kim D amp Fischer I 2000 Improved recombinant retroviral titers utilizing trichostatin A Biotech niques 29 4 884 890 Contact Us For Assistance Customer Service Ordering Technical Support Telephone 800 662 2566 toll free Telephone 800 662 2566 toll free Fax 800 424 1350 toll free Fax 650 424 1064 Web www clontech com Web www clontech com E mail orders clontech com
24. e widely accepted as the standard target cell for titrating lentivirus because these cells are transduced very efficiently Note that the same virus preparation can yield different apparent titers on different cells lines due to host cell factors that can produce very different transduction efficiencies Some variations of the drug resistance colony assay employ either a shorter selection period 3 days Byun et al 1996 recently infected target cells Tafuro et al 1996 Miyao et al 1995 or in situ PCR PRINS Claudio et al 2001 but achieve similar results Other methods for the direct quantitation of virus particles include slot blots Nelson ef 4 1998 Murdoch et al 1997 Onodera et al 1997 and PCR applied to viral supernatants Quinn amp Trevor 1997 Morgan et al 1990 Reverse transcriptase activity has also been used for titration Goff er 4 1981 www clontech com Protocol No PT5135 1 Version No PROY3745 15 Lenti X Lentiviral Expression Systems User Manual VIII Transducing Target Cells with Lenti X Viruses A Protocol Transducing Target Cells with Lenti X Viruses The following protocol is a general method for transducing adherent cell lines such as HT 1080 or HeLa using Protocol polybrene Polybrene is a polycation that reduces charge repulsion between the virus and the cellular membrane The a optimum final concentration of polybrene may be determined empirically but generally falls withi
25. eff ciency see Section VII B If you are concerned that exposure to either the polybrene or to the viral superna tant which contains medium conditioned by the packaging cells may adversely affect your target cells limit the transduction to 6 8 hr 6 Remove and discard the virus containing transduction medium and replace it with fresh growth medium Caution discarded medium contains infectious lentivirus 7 Continue to incubate the cells for 24 48 hr to allow your gene product to accumulate in the target cells 8 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 sa Using Untitrated Lenti X Virus Stocks and Supernatants The Lenti X HTX Packaging System is capable of producing very high virus titers Using excessive eo amounts of virus can be detrimental to target cell performance and viability If you have not deter Attention mined the titer of your virus stock perform transduction experiments using several different fold di lutions to test a range of MOls At Clontech our scientists can often transduce an entire 100 mm dish of target cells using 10 100 ul of unconcentrated Lenti X supernatant Protocol No PT5135 1 Version No
26. es C centrifugation and CH 296 coated plates effect of gene transfer protocol on T cell homing receptor expression Hum Gene Ther 12 15 1843 1855 Moritz T Dutt P Xiao X Carstanjen D Vik T Hanenberg H amp Williams D A 1996 Fibronectin improves transduction of reconstituting hematopoietic stem cells by retroviral vectors evidence of direct viral binding to chy motryptic carboxy terminal fragments Blood 88 3 855 862 Hanenberg H Xiao X L Dilloo D Hashino K Kato I amp Williams D A 1996 Colocalization of retrovi rus and target cells on specific fibronectin fragments increases genetic transduction of mammalian cells Nat Med 2 8 876 882 Bajaj B Lei P amp Andreadis S T 2001 High efficiencies of gene transfer with immobilized recombinant retrovirus kinetics and optimization Biotechnol Prog V7 4 587 596 G Cocultivation of target cells and packaging cells Allows targets to be continuously in con tact with freshly produced viral supernatant Casal M L amp Wolfe J H 1997 Amphotropic and ecotropic retroviral vector viruses transduce midgestational murine fetal liver cells in a dual chambered cocultivation system Gene Ther 4 1 39 44 Germeraad W T Asami N Fujimoto S Mazda O amp Katsura Y 1994 Efficient retrovirus mediated gene transduction into murine hematopoietic stem cells and long lasting expression using a transwell coculture system Bloo
27. ghts for uses other than in conjunction with this product or to use this product for purposes other than research please contact Open Biosystems licensing officer at 256 319 1462 Living Colors Products ACGFP1 AmCyan AsRed mBanana mCherry DsRed HcRed mOrange mPlum mRaspberry mStrawberry td Iomato ZsGreen ZsYellow and their variants Not For Profit Entities Orders may be placed in the normal manner by contacting your local representative or Clontech Customer Service at 650 919 7300 At its discretion Clontech grants Not For Profit Entities a non exclusive personal limited license to use this product for non commercial life science research use only Such license specifically excludes the right to sell or otherwise transfer this product its components or derivatives thereof to third parties No modifications to the protein coding seguence may be made without express written permission from Clontech Any other use of this product reguires a license from Clontech For license informa tion please contact a licensing representative by phone at 650 919 7320 or by e mail at licensing clontech com For Profit Entities wishing to use this product are reguired to obtain a license from Clontech For license information please contact a licensing representative by phone at 650 919 7320 or by e mail at licensing clontech com www clontech com Protocol No PT5135 1 Version No PROY3745 23 Lenti X Lentiviral Expression Systems User Manual
28. gma Aldrich Co No D5796 and 10 fetal bovine serum Add 1 mM sodium pyruvate Tetracycline free fetal bovine serum FBS see important information below We strongly recommend using Tet System Approved FBS Cat Nos 631101 amp 631106 for all packaging cell transfections and for cultur ing target cells when using a Lenti X Tet Advanced Inducible Expression System e Cell growth medium and supplies specific for your target cells o Attention PROY3745 Tetracycline Free Fetal Bovine Serum FBS for Packaging Cell and Target Cell Culture Many lots of bovine sera are contaminated with tetracycline Tc or its derivatives which can affect basal expression or inducibility in Tet Expression Systems Figure 3 It is critical that the FBS used for cell culture not interfere with Tet responsive expression e The Lenti X HTX Packaging Mix utilizes Tet Off transactivation to drive high level expression of specific viral packaging proteins The presence of Tc contaminants in FBS will reduce expression of these important components and will negatively affect viral titers Therefore 293T cells that host the Lenti X HTX Packaging System must be cultured in medium containing Tc free FBS e Tc contaminants in FBS will also significantly diminish the performance of the Tet On and Tet Off Advanced Systems in target cells e These problems can be eliminated by using a Tet System Approved FBS Cat Nos 631101 amp 631106 from
29. h the Clontech logo and all other trademarks are the property of Clontech Laboratories Inc unless noted otherwise Clontech is a Takara Bio Company 2010 Clontech Laboratories Inc Protocol No PT5135 1 www clontech com Clontech Laboratories Inc Version No PROY3745 ATakara Bio Company 24
30. igh efficiency transfection produces very high virus titers that can be as much as 25 50 times higher than other commercially available systems Lenti X supernatants can very often be used to infect target cells directly without prior concentration www clontech com Protocol No PT5135 1 Version No PROY3745 3 Lenti X Lentiviral Expression Systems User Manual l Introduction continued Protocol No PT5135 1 Version No 4 PROY3745 Core lentiviral vector backbone cPPT RRE CTS Vector specific elements Lentiviral Vectors for Constitutive cDNA Expression H Lentiviral Vectors for shRNA Expression pLVX Puro PLVX shRNA1 pA pA Powe Z Pook Puro Pos Pook Puro pLVX shRNA2 E Lentiviral Vectors for Bicistronic Expression PLVX IRES Puro Neo Hyg IRES Puro Neo H Ep W H Lentiviral Vectors for ProteoTuner Protein Control pLVX IRES FP pLVX PTuner n Powe S IRES IRES Puro FP fluorescent protein mCherry tdTomato or ZsGreen1 pLVX PTuner Green N Posy IE DD Lentiviral Vectors with an EF1 Promoter pLVX EF1 IRES Puro 1G Lentiviral Vectors for Fluorescently Tagged Protein Expression P IRES Puro EF1 pLVX FP N1 a l FP pA pLVX EF1 IRES FP P P CMV IE PGK Puro n Pa 4 IRES pLVX FP C1 FP fluorescent protei
31. ion Virus purification enables you to remove cellular contaminants that could otherwise adversely affect your transduction experiments Lenti X Maxi Purification Kits Cat Nos 631233 amp 631234 produce outstanding yields of highly purified virus from crude supernatants The gravity column based protocol is fast simple and effective and produces virus that is fully intact and fully functional E Lentivirus Concentration Use Lenti X Concentrator Cat Nos 631231 amp 631232 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 F Antibiotics for Selecting Transduced Cells G418 Cat No 631307 Puromycin Cat Nos 631305 amp 631306 Hygromycin B Cat No 631309 are used for performing drug selection of target cells transduced with Lenti X viruses having the respective resistance genes and for titrating the corresponding Lenti X virus stocks by drug selection Prior to using these antibiotics determine the optimal selection concentration for each cell type as described in Appendix A www clontech com Protocol No PT5135 1 Version No PROY3745 7 Lenti X Lentiviral Expression Systems User Manual ll Additional Materials Reguired continued Protocol No PT5135 1 Version No 8 PROY3745 G Polybrene for Viral Transductions Polybrene hexadimeth
32. ion Systems User Manual lll Safety Guidelines for Working with Lentiviruses o Attention Clontech Laboratories Inc ATakara Bio Company The protocols in this user manual require the production handling and storage of infectious lentivirus It is imperative to fully understand the potential hazards of and necessary precautions for the laboratory use of lentiviruses The National Institute of Health and Center for Disease Control have designated recombinant lentiviruses as Level 2 organisms This requires the maintenance of a Biosafety Level 2 facility for work involving this virus and others like it The pseudotyped lentiviruses packaged from the HIV 1 based vectors described here are capable of infecting human cells The viral supernatants produced by these lentiviral systems could depending on your insert contain potentially hazardous recombinant virus Similar vectors have been approved for human gene therapy trials attesting to their potential ability to express genes in vivo For these reasons due caution must be exercised in the production and handling of any recombinant lentivirus The user is strongly advised not to create pseudotyped lentiviruses capable of expressing known oncogenes For more information on Biosafety Level 2 agents and practices download the following reference e Biosafety in Microbiological and Biomedical Laboratories BMBL Fifth Edition February 2007 HHS Pub No CDC 93 83
33. ire 1200 pl of DNA Xfect solution from Step 5 dropwise to cultured cells from Step 1 Rock the plate gently back and forth to mix Note It is normal for the medium to change color slightly upon addition of the DNA Xfect solution 8 Incubate the plate at 37 C 9 After 4 hr to overnight replace the transfection medium with 10 ml fresh complete growth medium con taining Tet System Approved FBS and incubate at 37 C for an additional 24 48 hr Viral titers will gener ally be highest at 48 hr after the start of transfection Caution discarded medium contains infectious lentivirus 10 Harvest the lentiviral supernatants and pool similar stocks if desired Caution supernatants contain infectious lentivirus 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 polysulfonic low protein binding instead of nitrocellulose Nitrocellulose binds proteins present in the membrane of lentivirus and destroys the virus 11 Verify virus production with Lenti X GoStix see protocol PT5123 2 for details or titrate the virus stock Section VII then use the virus to transduce target cells or store at 80 C Note Titers can drop as much as 2 4 fold with each freeze thaw cycle Clontech Laboratories Inc ATakara Bio Company www clontech com Lenti X Lentiviral Expression Systems User Manual VII Determining Lentivi
34. iverse viral vector preparations using a simple and rapid whole virion dot blot method Hum Gene Ther 9 2401 2405 Onodera M Yachie A Nelson D M Welchlin H Morgan R A amp Blaese R M 1997 A simple and reliable method for screening retroviral producer clones without selectable markers Hum Gene Ther 8 1189 1194 Quinn T P amp Trevor K T 1997 Rapid quantitation of recombinant retrovirus produced by packaging cell clones Biotechniques 23 1038 1044 Rapid Lentiviral and Retroviral Titration Kits January 2008 Clontechniques XXIII 1 1 3 Sambrook J Fritsch E F amp Maniatis T eds 2001 Molecular Cloning A Laboratory Manual Cold Spring Harbor Laboratory Press Cold Spring Harbor NY Tafuro S Zentilin L Falaschi A amp Giacca M 1996 Rapid retrovirus titration using competitive polymerase chain reaction Gene Ther 3 679 684 Transfection Reagents for High Titer Lentivirus October 2007 Clontechniques XXII 4 8 Wu X Wakefield J K Liu H Xiao H Kralovics R Prchal J T amp Kappes J C 2000 Development of a Novel Trans Lentiviral Vector That Affords Predict able Safety Mol Ther 2 47 55 Yao F Svenjo T Winkler T Lu M Eriksson C amp Eriksson E 1998 Tetracycline repressor tetR rather than the tetR mammalian cell transcription factor fu sion derivatives regulates inducible gene expression in mammalian cells Hum Gene Ther 9 1939 1950 Zennou
35. ll cells within 3 4 days e Lot to lot variations in potency exist for all selection drugs so each new lot of antibiotic should be titrated 1 For each antibiotic to be tested plate 2 x 10 cells in each well of a 6 well plate containing 3 ml of the appro priate complete medium plus increasing concentrations of G418 0 50 100 200 400 and 800 pg ml For puromycin add the drug at 0 1 0 2 5 5 0 7 5 and 10 0 pg ml 2 For G418 incubate the cells for 5 10 days or until all cells are dead Examine the dishes for viable cells every two days Replace the selective medium every four days or more often if necessary until the optimal con centration is determined 3 For puromycin incubate the cells 4 7 days Replace medium after 2 days to remove dead cells B Protocol Concentrating Virus Using Ultracentrifugation Note Lenti X Concentrator Cat Nos 631231 amp 631232 is a very cost effective reagent that allows fast simple and highly efficient concentration of any lentiviral stock without using ultracentrifugation In the simple protocol lentivi ral supernatant is mixed with the Lenti X Concentrator reagent incubated for a short period and spun in a standard centrifuge This ultracentrifugation protocol should be used for VSV G enveloped virions only Burns er al 1994 1 Remove cell debris and aggregated virus by low speed centrifugation 500 x g for 10 min at 47C 2 Pellet the virus at 50 000 x g for 90 min at 4
36. may be weak 2 Low expression of GOI or possibly inactivated in Insert a tissue specific promoter for GOI expression target cells Poor target cell viability Check growth parameters MOI too high i e too much i Z 10 go high li too Dilute virus stock titrate the virus virus used N Reduce or optimiz lybrene concentration Polybrene toxicity a a au ie ee re re 3 Infection is toxic to target PES UCe IMIECHON ELUTE cells Dilute virus stock using target cell culture medium harvest virus from packaging cells using target cell medium Consider purifying your virus prior to transduction using the Lenti X Maxi Purification Kit Cat Nos 631233 amp 631234 Viral supernatant contains transduction inhibitors Packaging cell supernatant or medium is toxic to cells Protocol No PT5135 1 www clontech com Clontech Laboratories Inc Version No PROY3745 ATakara Bio Company 18 Lenti X Lentiviral Expression Systems User Manual X 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 Byun J Kim J M Kim S H Yim J Robbins P D amp Kim S 1996 A simple and rapid method for the determination of recombinant retrovirus titer by G418 selection Gene Ther 3 1018 1020 Claudio P P Cinti C amp Giordano A 2001 Application of the primer in situ DNA synthesis P
37. mouse and rat cells to be transduced with high efficiency The lentiviral supernatants produced by the transfected packaging cells can then be used to infect and transduce target cells to express your GOI fusion protein or shRNA Clontech has developed several highly advanced Lenti X expression systems that provide the broad cellular tropisms of pseudotyped lentivirus very high titers of safe nonreplicating virus and excellent transgene expression levels Clontechniques October 2007 B Lenti X Vectors Clontech offers Lenti X vectors for many applications Figure 1 All pLVX vectors possess the requisite HIV 1 LTRs and the lentiviral packaging signal V as well as other elements to improve transgene expression viral titer and overall vector function e WPRE A woodchuck hepatitis virus posttranscriptional regulatory element prevents poly A site readthrough promotes RNA processing and maturation and increases nuclear export of RNA Zufferey et al 1999 Higashimoto et al 2007 It works in the context of viral genomic transcripts in packaging cells to enhance vector packaging and increase the viral titers In addition the WPRE boosts expression of your GOI in transduced target cells by facilitating the maturation of mRNA transcripts produced by the vector s internal promoter e g Pomy or Prend MV Tight e cPPT CTS A central polypurine tract central termination sequence creates a DNA flap that increases nuclear importation of
38. n mCherry or ZsGreen1 oO Pom te S Pog Puro pLVX EF1 IRES FP N1 FP fluorescent protein AcGFP1 AmCyan1 DsRed Express2 DsRed Monomer mCherry tdTomato or ZsGreen1 Puro f pLVX EF1 IRES FP C1 E Lentiviral Vectors for Gene Expression Reporter Systems LVX MetLuc FP fluorescent protein AcGFP1 DsRed Monomer or mCherry pLVX DD_FP Py Lentiviral Vectors for Inducible cDNA Expression pLVX Tet On Advanced FP fluorescent protein AmCyan1 ZsGreen1 or tdTomato Pomvie yw IRES Neo pLVX Tet Off Advanced Posy te yN IRES Neo pLVX Tight Puro Tight Puro Vectors available as part of an expression system Vectors available separately Figure 1 Lentiviral vectors for many applications Lenti X vectors contain the LTRs packaging signal Rev response element RRE and central polypurine tract central termination sequence cPPT CTS from HIV 1 and include a wood chuck hepatitis virus posttranscriptional regulatory element WPRE See text for descriptions All vectors are designed to be used with a Lenti X HTX Packaging System and the Lenti X 293T Cell Line which together produce very high titers of pseudotyped lentivirus for transducing virtually any cell type DD degradation domain IRES internal ribosome entry sequence FP fluorescent protein MCS multiple cloning site MetLuc Metridia luciferase P cytomegalovirus immedi a
39. n a range of 2 12 y ug ml However excessive exposure to polybrene gt 24 hr can be toxic to cells This protocol can be used as a starting point for determining the optimal transduction conditions for your target cells Refer to Appendix B for additional references and alternative infection methods For cells that are difficult to transduce or that might be sensitive to polybrene RetroNectin Reagent Takara Bio USA Cat Nos TAK 100A amp 100B can be used to greatly improve transduction efficiency 1 Plate target cells in their complete growth medium 12 18 hr before transduction 2 Thaw aliquots of your cleared and titrated lentiviral stock or use cleared virus stock freshly prepared from packaging cells Section VI Mix gently but do not vortex Note that each freeze thaw cycle will decrease titer by 2 4 fold 3 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 pg ml 4 Dilute the lentiviral stock with medium to obtain the desired MOI 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 See Information Box below 5 Add viral supernatant to the cells and transduce for 8 24 hr Centrifuge the cultures to improve infection
40. natant Figure 2 Lentivirus production with the Lenti X HTX Packaging System and Lenti X 293T cells Initially cotransfection of a Lenti X vector and the Lenti X HTX Packaging Mix Step 1 results in the production of the corresponding recombi nant lentiviral genomic RNA transcript and viral packaging proteins Step 2 A vector in the packaging mix encodes the Tet Off transactivator tTA which produces extra high expression of specific packaging proteins via Tet Off transactiva tion Recognition of the packaging sequence on the recombinant viral RNA genome by the packaging proteins Step 3 results in the assembly of viral cores which are transported to the cell membrane Step 4 Cores are then enveloped by cellular membrane containing aggregated VSV G or ecotropic gp70 envelope proteins Mature infectious virions then bud from the cell Step 5 and are collected in the medium Step 6 While the virions are infectious they lack sev eral critical genes required for their subsequent replication and production in target cells The use of multiple plasmids with which to express the viral proteins 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 www clontech com Protocol No PT5135 1 Version No PROY3745 5 Lenti X Lentiviral Expression Systems User Manual Protocol No PT5135 1 Version No 6
41. nology or its component parts to third parties Notwithstanding the above academic and not for profit research institutions whose research using the Tet Technology is sponsored by for profit organizations which shall receive ownership to all data and results stemming from the sponsored research shall need a commercial license agreement from TET Systems in order to use the Tet Technology In accepting this license all users acknowledge that the Tet Technology is experimental in nature TET Systems GmbH amp Co KG makes no warranties express or implied or of any kind and hereby disclaims any warranties representations or guarantees of any kind as to the Tet Technology patents or products All others are invited to request a license from TET Systems GmbH amp Co KG prior to purchasing these reagents or using them for any purpose Clontech is required by its licensing agreement to submit a report of all purchasers of the Tet controllable expression system to TET Systems For license information please contact GSF CEO TET Systems GmbH amp Co KG Im Neuenheimer Feld 582 69120 Heidelberg Germany Tel 4962215880400 Fax 4962215880404 eMail info tetsystems com or use the electronic licensing request form via http www tetsystems com main_inquiry htm Transfection Polymers Use of this product is covered by one or more of the following U S Patent Nos and corresponding patent claims outside the US 6 998 115 7 427 394 This product is intended fo
42. nti X Packaging System see Section II B Perform all steps in asterile tissue culture hood Lentivirus requires the use of a Biosafety Level 2 facility Recombinant pseudotyped lentiviruses packaged from HIV 1 based vectors are capable of infecting human cells Know and use appropriate safety precautions See Section III 1 Approximately 24 hr before transfection seed 4 5 x 10 Lenti X 293T 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 Con tinue 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 2 Thoroughly vortex Xfect Polymer 3 For each transfection sample prepare two microcentrifuge tubes by adding reagents in the order listed Tube 1 Plasmid DNA Tube 2 Polymer 557 ul Xfect Reaction Buffer 592 5 pl Xfect Reaction Buffer _36 pl Lenti X HTX Packaging Mix _ 7 5 pl Xfect Polymer __7 ul Lenti X Vector DNA 1 ng nl 600 pl Total Volume 600 pl Total Volume Note It is crucial that the Xfect Polymer does not remain in aqueous solution for longer than 30 min at room temperature 4 Vortex each tube well to mix 5 Add the Polymer solution Tube 2 to the DNA solution Tube 1 and vortex well at a medium speed for 10 sec 6 Incubate each DNA Xfect mixture for 10 min at room temperature to allow nanoparticle complexes to form 7 Add the ent
43. nviverevecgacuessudesaced seh wa vaviwuuus E E 19 Appendix A Additional Protocols cccccecceceeeeeeeeeeeeeeeeeeeeeeeeeeeaeaaaeaeeeeeeseeeeseseeesseaaneneeeees 20 A Protocol Titrating Antibiotics for Selecting Stable Cell Lines oo i eceseeessecesesenseeeeeeeseeeeeeerseeeneeanes 20 B Protocol Concentrating Virus Using Ultracentrifugation ote eseesseeecseseeeseeseseeesesesenseetecneneeeeee 20 Appendix B Additional Viral Infection Methods c cccccesseceeeeesceeeeeeesseeeeeeessneeeeeeessnaaes 21 List of Figures Figure 1 Lentiviral vectors for many applications ccsessesessssseeseseeeseescsesesesecsesesnsecscaeanssecseasenseeseaseneeeenes 4 Figure 2 Lentivirus production with the Lenti X HTX Packaging System and Lenti X 293T cells 2 Figure 3 Tetracycline activity in bovine sera wo cccssesescseseseeeseeseseeecesescsseceecscaesececesseeesesasscsseaeessenseesesanaes 6 Figure 4 Advanced features of NucleoBond Xtra Maxi and Midi Columns and NucleoBond Finalizer 10 Figure 5 The Lenti X GoStix protocol takes only 30 seconds seeessesssseeseesteeeceesesessesesesenseeeecneaeeeeee 13 List of Tables Table I Troubleshooting Guide for Lenti X Expression Systems ccccsscssesssssseeeseeseeeeeesesesenseseesseaeeeees 17 Table II Recommended Concentrations for Selection Antibiotics ug ml oe ceeeeseseeseeeeseeeeeeeeeeneeeees 20 Protocol No PT5135 1 www clontech com Clontech Laboratories Inc Ve
44. op 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 pre warmed medium Mix gently 3 Slowly add an additional 4 ml of fresh pre warmed medium to the tube and mix gently 4 Add an additional 5 ml of pre warmed 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 Gently 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 Com plete 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 you should prepare frozen aliquots to provide a renewable source of cells Consult the Lenti X 293T Cell Line Protocol at a Glance PT 4058 2 for a cell freezing protocol Clontech Laboratories In
45. or stable transductants using the appropriate antibiotic Titers are calculated from the number of drug resistant colonies that develop after selection is completed www clontech com Protocol No PT5135 1 Version No PROY3745 13 Lenti X Lentiviral Expression Systems User Manual VII Determining Lentiviral Titer continued Protocol 7 14 days ok o Attention Protocol No PT5135 1 Version No PROY3745 14 B Protocol Determining Viral Titer Using Antibiotic Selection l Plate HT 1080 cells or another cell line in one 6 well plate the day before performing the titration infec tions Plate 2 x 10 cells well in 2 ml of medium Reserve at least one well for a no infection control Prepare 20 ml of complete medium and add 60 pl of 4 mg ml polybrene This concentration of polybrene 12 pg ml will be eventually diluted 3 fold for a final concentration of 4 pg 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 pg ml Excessive exposure to polybrene gt 24 hr can be toxic to cells Prepare cleared viral supernatant from the transfected Lenti X 293T packaging cells Section VI 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
46. polybrene from Step 2 to each of six sterile and numbered 1 5 ml microfuge tubes b Add 150 pl of the virus stock from Step 3 to tube 1 Mix gently c Transfer 150 pl from tube 1 to tube 2 and mix Continue making serial dilutions by transferring 150 pl from each successive dilution into the next prepared tube Infect the HT 1080 cells by adding 1 ml from each of the 5 least concentrated viral dilutions Step 4 to the appropriately labeled wells The final polybrene concentration will be 4 pg ml in 3 ml Centrifuge the cultures to improve transduction efficiency After infecting for 8 24 hours remove the supernatants and begin antibiotic selection using the concentra tion of antibiotic that is optimal for your cell line Appendix A Caution discarded medium contains infectious lentivirus Allow drug resistant colonies to form for 7 14 days Stain the colonies with 1 crystal violet solution in 10 ethanol and count The titer of the virus stock corresponds to the number of colonies generated by the least concentrated dilu tion 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 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 i
47. r research purposes only It may not be used for i any human or veterinary use including without limitation therapeutic and prophylactic use ii any clinical use including without limitation diagnostic use iii screening of chemical and or biological compounds for the identification of pharmaceutically active agents including but not limited to screening of small molecules target validation preclinical testing services or drug development Any use of this product for any of the abovementioned purposes requires a license from the Massachusetts Institute of Technology VSV G Technology VSV G is licensed from Pangenix and its use is covered under U S Patent Nos 5 512 421 and 5 670 354 Rights to use this product are limited to nonhuman re search only and use of the patented technology in domestic ungulates is expressly prohibited No other rights are conveyed Inquiry into the availability of a license for commercial purposes should be directed to Jane C Burns M D Pangenix 6505 El Camino del Teatro La Jolla CA 92037 WPRE Technology Clontech has a license to sell products containing WPRE under the terms described below Any use of WPRE outside of Clontech s product or the products intended use reguires a license as detailed below Before using the product containing WPRE please read the following license agreement If you do not agree to be bound by its terms contact Clontech within 10 days for authorization to return the unused prod
48. r virus production is within a usable range or for selecting the best time to harvest your virus A 3 prep sample is supplied for free with many of Clontech s Lenti X systems Coonteen 9 p Conen gt 5 x 10 IFU mI Figure 5 The Lenti X GoStix protocol takes only 30 seconds Clontech Laboratories Inc ATakara Bio Company qRT PCR Clontech offers a convenient Lenti X qRT PCR Titration Kit Cat No 632165 for rapid titra tion of lentiviral supernatants It employs One Step qRT PCR and SYBR Green chemistry in a 4 hr protocol that can be used with any lentiviral vector regardless of the marker involved and is beneficial for comparing the titers of different vectors and for titrating freshly harvested virus stocks p24 ELISA The Lenti X p24 Rapid Titer Kit Cat No 632200 uses ELISA to specifically measure the amount of p24 capsid protein present in your viral supernatant and then correlates the level of p24 to virus titer The assay requires 4 hr to complete Flow cytometry For Lenti X vectors containing a fluorescent marker cells can be transduced using the protocol in Section 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 Lenti X vectors that contain a selectable marker cells are infected with serial dilu tions of the virus stock and then selected f
49. ral Titer A Introduction To produce consistent transduction results using a known multiplicity of infection MOD it is necessary to titrate your Lenti X virus 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 Titer values will depend heavily on the cell type and method used for titration so there may be significant differences between titers determined in cells typically used for titration e g HT 1080 and the number of target cells that are ultimately transduced However 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 ofa selectable or fluorescent marker Lenti X titration kits can be used with any HIV 1 based lentiviral vector Instant Lentivirus Test You can assess the quality of your lentivirus stock in 30 seconds with Clontech s Lenti X GoStix Cat Nos 631241 631243 amp 631244 The GoStix detect lentiviral p24 in only 20 pl and can be used to determine whethe
50. rine bromide Sigma Aldrich No H9268 is needed for the standard infection transduction protocol to facilitate lentiviral gene transfer Polybrene is a polycation that reduces charge repulsion between the virus and the cellular membrane The optimal polybrene concentration for your target cells maximal infectivity with mini mal toxicity should be determined empirically by testing concentrations in the range of 2 12 pg ml For cells that are especially sensitive to polybrene consider using RetroNectin Reagent H RetroNectin Reagent for Enhanced Viral Transductions RetroNectin Reagent available from Clontech Laboratories Inc or Takara Bio USA Cat Nos TAK 100A TAK 100B is a recombinant fragment of fibronectin CH 296 that can be used to greatly improve retroviral and lentiviral transduction efficiencies Clontechniques October 2008 RetroNectin is coated onto tissue culture plates to provide a substratum that binds both viruses and cells The colocalization of virus and cells on this novel substratum improves cell virus contact and enhances transduction This is especially useful for cells grown in suspension e g lymphocytes and lymphocyte cell lines and other cells that are difficult to transduce such as hematopoietic stem cells or for cells that may be especially sensitive to polybrene Visit www takarabiousa com for more information www clontech com Clontech Laboratories Inc ATakara Bio Company Lenti X Lentiviral Express
51. rsion No PROY3745 ATakara Bio Company 2 Lenti X Lentiviral Expression Systems User Manual l Introduction Clontech Laboratories Inc ATakara Bio Company A Gene Transfer and Expression Using Recombinant Lentiviruses Recombinant lentiviral vectors are powerful and efficient tools for transferring heritable genetic material into the ge nome of virtually any cell type Ausubel er al 1995 Coffin et al 1996 Lentiviruses are perhaps the most versatile of retroviruses since they are able to infect transduce and sustain expression in almost any mammalian cell including dividing and nondividing cells stem cells and primary cell cultures In the Lenti X systems high titers of recombinant replication incompetentvirionsare easily generated using a Lenti X HTX Packaging System Cat Nos 631247 631249 or 631251 in which a Lenti X expression vector Figure 1 containing your gene of interest GOI is cotransfected along with a Lenti X HTX Packaging Mix into the Lenti X 293T Cell Line Cat No 632180 The Lenti X HTX Packaging Mix and the Lenti X HTX Ecotropic Packaging Mix are optimized mixtures of plasmids that respectively express viral proteins in ratios that have been optimized for high efficiency packaging of lentiviral vector transcripts into either VSV G or ecotropically pseudotyped lentiviral particles The VSV G envelope protein works for almost any cell type while the ecotropic envelope glycoprotein gp70 from MLV enables
52. s not available www clontech com Clontech Laboratories Inc ATakara Bio Company Lenti X Lentiviral Expression Systems User Manual VII Determining Lentiviral Titer continued Clontech Laboratories Inc ATakara Bio Company C Alternative Titration Methods The Lenti X qRT PCR Titration Kit directly quantitates the viral genomes in your virus stock which is much faster and more versatile than antibiotic selection Since it does not rely on antibiotic selection all par ticles regardless of genome sequence or infectivity can be quantitated Functional titers do not yield accurate measures of virion concentration and are subject to the infection and transduction efficiencies of the cell line being used for titration The Lenti X p24 Rapid Titer Kit employs a straightforward ELISA of the HIV 1 p24 capsid protein to mea sure lentiviral titer p24 content is correlated to virus content either by comparing p24 content to a superna tant of known titer or by calculation You may also determine viral titer by infecting HT 1080 cells with serially diluted viral supernatants pro duced using a control vector containing an easily detectable reporter gene e g LacZ luciferase or a fluores cent protein Test virus infection on both HT 1080 cells and your target cells Infecting your target cell line will give you a rough but rapid estimation of infection success You can use other cell lines to determine viral titer but HT 1080 cells ar
53. te early promoter enhancer P o phosphoglucokinase promoter Paaie the modified Tet responsive promoter P human U6 snRNA promoter RNA Pol lll www clontech com Clontech Laboratories Inc ATakara Bio Company Lenti X Lentiviral Expression Systems User Manual l Introduction continued V e Attention Clontech Laboratories Inc ATakara Bio Company C Lenti X HTX Packaging Systems cont d Highest Safety For added biosafety the genes that express the viral packaging proteins have been split onto dif ferent plasmids to prevent the collective inclusion of these coding sequences into viral particles during the pack aging process The lack of sequence homology between the packaging mix plasmids and our Lenti X Vectors also prevents transfer via homologous recombination This split gene trans expression strategy effectively prevents the production of replication competent lentivirus e g the viruses cannot replicate autonomously in target cells Lenti X HTX Packaging Mix QA 1 Cotransfection of vector and OC Lenti X HTX Packaging Mix Xfect transfection oS en eu 4 Transcription Lenti X Transient and translation vector expression N Viral 472 7 proteins NT Lenti X 293T 3 Viral proteins y Gol MI recognize RNA Packaging Cell 4 Assembly of virus cores ii 5 Budding of infectious O virions O 6 Harvest lentivirus in culture super
54. ted gene transfer into T cells 95 transduction efficiency without further in vitro selection Hum Gene Ther 11 8 1189 1200 Bahnson A B Dunigan J T Baysal B E Mohney T Atchison R W Nimgaonkar M T Ball E D amp Barranger J A 1995 Centrifugal enhancement of retroviral mediated gene transfer J Virol Methods 54 2 3 131 143 C Precipitation of virus to increase titer concentration Pham L Ye H Cosset F L Russell S J amp Peng K W 2001 Concentration of viral vectors by coprecipitation with calcium phosphate J Gene Med 3 2 188 194 Darling D Hughes C Galea Lauri J Gaken J Trayner I D Kuiper M amp Farzaneh F 2000 Low speed centrifugation of retroviral vectors absorbed to a particulate substrate a highly effective means of enhancing retroviral titre Gene Ther 7 11 914 923 Hughes C Galea Lauri J Farzaneh E amp Darling D 2001 Streptavidin paramagnetic particles provide a choice of three affinity based capture and magnetic concentration strategies for retroviral vectors Mol Ther 3 4 623 630 www clontech com Protocol No PT5135 1 Version No PROY3745 21 Lenti X Lentiviral Expression Systems User Manual Appendix B Additional Viral Infection Methods continued Protocol No PT5135 1 Version No 22 PROY3745 D Precipitation during transduction facilitates greater contact between the target cells and virions Le Doux
55. tiple cloning site MCS Consult the Vector Information Packet provided with each Lenti X vector for additional cloning de tails You can also use the In Fusion HD Cloning System Cat No 639645 which allows PCR products to be easily cloned into any linearized vector Note Depending on the Lenti 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 im prove 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 recombi nant virions Coffin et al 1997 4 Perform a midi or maxi scale plasmid DNA preparation for each plasmid that will be transfected into the packaging cells For guaranteed transfection grade plasmid DNA we recommend using NucleoBond Xtra Midi Plus or Maxi Plus Kits Figure 4 Cat Nos 740412 10 and 740416 10 For rapid production of endotoxin free transfection grade plasmid DNA use NucleoBond Xtra Midi EF Plus or Maxi EF Plus Kits Cat Nos 740422 10 and 740426 10 xX A New column filter lt Fast filtration NucleoBond Finalizer for fast DNA precipitation Improved silica material High binding capacity EE NucteoBond
56. uct containing WPRE and to receive a full credit Patents The WPRE technology is covered by one or more of the following U S Patents and corresponding patent claims outside the U S 6 136 597 6 284 469 6 312 912 6 287 814 issued to The Salk Institute for Biological Studies Individual License Agreement Clontech grants you a non exclusive license to use the enclosed product containing WPRE in its entirety for its intended use The product is being transferred to you in furtherance of and reliance on such license Any use of WPRE outside of Clontech s product or the product s intended use requires a license from the Salk Institute for Biological Studies Termination of License This license agreement is effective until terminated You may terminate it at any time by destroying all products containing WPRE in your control It will also terminate automatically if you fail to comply with the terms and conditions of the license agreement You shall upon termination of the license agreement destroy all products containing WPRE in your control and so notify Clontech in writing This License shall be governed in its interpretation and enforcement by the laws of the State of California Contact for WPRE Licensing The Salk Institute for Biological Studies 10010 North Torrey Pines Road La Jolla CA 92037 Attn Office of Technology Management Phone 858 453 4100 ext 1275 Fax 858 546 8093 Attn Office of Technology Management Clontec
57. urchase new aliquot of Lenti X 293T cells C Virus Production Plate 4 5 x 10 cells 100 mm plate or fewer if the cells di vide rapidly Use at 50 80 confluency See Section VI Improper thawing techniques Cells plated too densely 1 Poor transfection efficiency as determined by GOI or ae MM MU Lenti X 293T cell line Cells harvested or analyzed Wait 48 hr after transfection for maximal expression Serum in medium contains Use Tet System Approved FBS Cat Nos 631101 amp tetracycline contaminants 631106 in the 293T culture medium See above section Concentrate the virus using centrif ugation see Appendix A or use the Lenti X Concentra tor Cat Nos 631231 amp 631232 to increase your avail able titer up to 100 fold without ultracentrifugation Virus harvested too early Harvest virus 48 72 hr after the start of transfection 2 Low titers lt 10 cfu ml Vector too large The limit of packaging function is 9 7 kb from 5 LTR to 3 LTR Polybrene missing or at Add polybrene 4 ug ml during transduction or suboptimal concentration optimize the concentration 2 12 ug ml Virus exposed to multiple Each cycle reduces titer by approximately 2 4 fold freeze thaw cycles Limit the number of freeze thaws Suboptimal selection proce Perform an antibiotic kill curve on the cell line prior to dure during titration using it for titration www clontech com Protocol No PT5135 1 Version No PROY3745 17
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