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ViraPower™ Lentiviral Expression Systems

1. Overview Introduction ViraPower HiPerform Lentiviral Expression Vectors Introduction TM TM The new ViraPower HiPerform Lentiviral Expression Systems allow the creation of a replication incompetent HIV 1 based lentivirus that is used to deliver and express your gene of interest in either dividing or non dividing mammalian cells The new expressions Systems use four new expression vectors two pLenti Gateway Destination vectors that are adapted for use with the Gateway technology pLenti6 3 V5 DEST Gateway vector and pLenti7 3 V5 DEST Gateway vector and two pLenti TOPO vectors that combine the ViraPower HiPerform Lentiviral Expression Systems with the rapid TOPO Cloning technology pLenti6 3 V5 TOPO vector and pLenti7 3 V5 TOPO vector For more information on these new vectors refer to the section below TM TM The new ViraPower HiPerform Lentiviral Expression vectors contain two new elements WPRE and cPPT to yield cell specific high performance results The WPRE Woodchuck Posttranscriptional Regulatory Element from the woodchuck hepatitis virus is placed directly downstream of the gene of interest allowing for increased transgene expression Zufferey et al 1998 with more cells expressing your gene of interest cPPT Polypurine Tract from the HIV 1 integrase gene increases the copy number of lentivirus integrating into the host genome Park 2001 and allows for a two fo
2. Antibiotic The pLenti6 3 expression constructs contain the Blasticidin resistance gene bsd Selection Kimura et al 1994 to allow for Blasticidin selection Takeuchi et al 1958 Yamaguchi et al 1965 of mammalian cells that have stably transduced the lentiviral construct TM TM Blasticidin is supplied with the ViraPower HiPerform Lentiviral Expression Kit but you can also purchase Blasticidin separately from Invitrogen page viii Preparing For more information about how to prepare and handle Blasticidin refer to the Blasticidin Appendix page 33 Continued on next page 22 Titering Your Lentiviral Stock Using Blasticidin Continued Determining Antibiotic Sensitivity Materials Needed Since you will be selecting for stably transduced cells using Blasticidin you must first determine the minimum concentration of Blasticidin required to kill your untransduced mammalian cell line i e perform a kill curve experiment Typically concentrations ranging from 2 10 pg ml Blasticidin are sufficient to kill most untransduced mammalian cell lines We recommend that you test a range of concentrations see protocol below to ensure that you determine the minimum concentration necessary for your cell line 1 Plate cells at approximately 25 confluence Prepare a set of 6 7 plates Allow cells to adhere overnight The next day substitute culture medium with medium containing varying concentrations of Blasticid
3. Note Expression of the VSV G glycoprotein causes 293FT cells to fuse resulting in the appearance of large multinucleated cells known as syncytia This morphological change is normal and does not affect production of the lentivirus 7 Post transfection Day 5 or 6 harvest virus containing supernatants by removing and transferring the medium into a 15 ml sterile capped conical tube Caution Remember that you are working with infectious virus at this stage Follow recommended guidelines for working with BL 2 organisms refer to page 5 8 Centrifuge supernatants at 2 000 x g for 15 minutes at 4 C to pellet debris 9 Optional Filter the viral supernatants through a Millex HV 0 45 um or equivalent PVDF filter see Note page 15 10 Pipet viral supernatants into cryovials in 1 ml aliquots 11 Store viral stocks at 80 C Proceed to Titering Your Lentiviral Stock page 16 Continued on next page 13 Producing Lentivirus in 293FT Cells Continued Reverse If you are an experienced user you may use the rapid reverse transfection Transfection procedure to cotransfect 293FT cells For information on positive controls see Procedure page 8 We recommend including a negative control no DNA no Lipofectamine 2000 in your experiment to help you evaluate your results You will need 6 x 10 293FT cells for each sample 1 On Day 1 prepare DNA Lipofectamine 2000 complexes for each transfection sample as follows a
4. pUC origin of replication ori Permits high copy replication and maintenance in E coli 37 Map and Features of pLP VSVG pLP VSVG Map 38 The figure below shows the features of the pLP VSVG vector The complete sequence of pLP VSVG is available for downloading from www invitrogen com or by contacting Technical Support see page 40 Comments for pLP VSVG 5821 nucleotides CMV promoter bases 1 747 TATA box bases 648 651 Human globin intron bases 880 1320 VSV G glycoprotein VSV G bases 1346 2881 Human globin polyadenylation signal bases 3004 3769 pUC origin bases 3927 4600 C Ampicillin bla resistance gene bases 4745 5605 C bla promoter bases 5606 5704 C C complementary strand Continued on next page Map and Features of pLP VSVG Continued Features of PLP VSVG pLP VSVG 5821 bp contains the following elements Features have been functionally tested Feature Benefit Human CMV promoter Permits high level expression of the VSV G gene in mammalian cells Andersson et al 1989 Boshart et al 1985 Nelson et al 1987 Human globin intron Enhances expression of the VSV G gene in mammalian cells VSV G glycoprotein VSV G Encodes the envelope G glycoprotein from Vesicular Stomatitis Virus to allow production of a pseudotyped retrovirus with a broad host range Burns et al 1993 Emi et al 1991 Yee et al 1994 Human gl
5. 2001 Lentivirus and foamy virus vectors novel gene therapy tools Expert Opinion on Biological Therapy 1 17 40 Park F and Kay MA 2001 Modified HIV 1 based lentiviral vectors have an effect on viral transduction efficiency and gene expression in vitro and in vivo Mol Ther 4 3 164 173 Sastry L Johnson T Hobson M J Smucker B and Cornetta K 2002 Titering Lentiviral vectors comparison of DNA RNA and marker expression methods Gene Ther 9 1155 1162 Continued on next page 48 References Continued Takeuchi S Hirayama K Ueda K Sakai H and Yonehara H 1958 Blasticidin S A New Antibiotic The Journal of Antibiotics Series A 11 1 5 White S M Renda M Nam N Y Klimatcheva E Y Zhu Fisk J Halterman M Rimel B J Federoff H Pandya S Rosenblatt J D and Planelles V 1999 Lentivirus vectors using human and simian imunodeficiency virus elements J Virology 73 2832 2840 Yamaguchi H Yamamoto C and Tanaka N 1965 Inhibition of Protein Synthesis by Blasticidin S I Studies with Cell free Systems from Bacterial and Mammalian Cells J Biochem Tokyo 57 667 677 Yee J K Miyanohara A LaPorte P Bouic K Burns J C and Friedmann T 1994 A General Method for the Generation of High Titer Pantropic Retroviral Vectors Highly Efficient Infection of Primary Hepatocytes Proc Natl Acad Sci USA 91 9564 9568 Yee J K 1999 in The Development
6. C TM The ViraPower Packaging Mix contains 3 tubes with 195 pg DNA per tube Upon receipt store at 20 C The following reagents are included with the One Shot Stb13 Chemically Competent E coli kit Transformation efficiency is 2 1 x 10 cfu ug plasmid DNA Store at 80 C Reagent Composition Qunatity S O C Medium 2 Tryptone 6ml 0 5 Yeast Extract 10 mM NaCl 2 5 mM KCI 10 mM MgCl 10 mM MgSO 20 mM glucose Stb13 Cells 21x 50 nl pUC19 Control DNA 10 pg ul in 5 mM Tris HCl 0 5 mM 50 pl EDTA pH 8 F mcrB mrr hsdS20 rs ms recA13 supE44 ara 14 galK2 lacY1 proA2 rpsL20 Str xyl 5 A leu mtl 1 Note This strain is endA1 TM TM Each ViraPower HiPerform Lentiviral Expression Kit includes the 293FT producer cell line The 293FT Cell Line is supplied as one vial containing 3 x 10 frozen cells in 1 ml of Freezing Medium Upon receipt store in liquid nitrogen For instructions to thaw culture and maintain the 293FT Cell Line see the 293FT Cell Line manual included with the ViraPower HiPerform Lentiviral Expression Kit To download the manual visit our website at www invitrogen com or contact Technical Support page 40 vii Accessory Products Introduction Additional Products viii The products listed in this section may be used with the ViraPower HiPerform Lentiviral Expression Kits For more information visit
7. Ina sterile 5 ml tube dilute 9 ug of the ViraPower Packaging Mix and 3 ug of pLenti expression plasmid DNA 12 ug total in 1 5 ml of Opti MEM I Medium without serum Mix gently b Ina separate sterile 5 ml tube dilute 36 ul Lipofectamine 2000 mix gently before use in 1 5 ml of Opti MEM I Medium without serum Mix gently and incubate for 5 minutes at room temperature c After incubation combine the diluted DNA Step a with the diluted Lipofectamine 2000 Step b Mix gently d Incubate for 20 minutes at room temperature to allow the DNA Lipofectamine 2000 complexes to form The solution may appear cloudy but this will not impede the transfection 2 While DNA lipid complexes are forming trypsinize and count the 293FT cells Resuspend the cells at a density of 1 2 x 10 cells ml in growth medium or Opti MEM I Medium containing serum Do not include antibiotics in the medium 3 Add the DNA Lipofectamine 2000 complexes Step 1d to a 10 cm tissue culture plate containing 5 ml of growth medium or Opti MEM I Medium containing serum Do not include antibiotics in the medium 4 Add 5 ml of the 293FT cell suspension from Step 2 6 x 106 total cells to the plate containing media and DNA Lipofectamine 2000 complexes Step 3 Mix gently by rocking the plate back and forth Incubate cells overnight at 37 C in a humidified 596 CO incubator 5 The next day Day 2 remove and discard the medium containing th
8. manufacture of such product If the purchaser is not willing to accept the limitations of this limited use statement Invitrogen is willing to accept return of the product with a full refund For information on purchasing a license to this product for purposes other than research contact Licensing Department Invitrogen Corporation 1600 Faraday Avenue Carlsbad California 92008 Phone 760 603 7200 Fax 760 602 6500 Continued on next page Purchaser Notification Continued Limited Use Label License No 109 Retroviral Helper Lines Retroviral helper cell lines are licensed from Wisconsin Alumni Research Foundation under U S Patent No 5 124 263 and corresponding patents and applications in other countries for internal research purposes only Use of these cell lines for Commercial Purposes requires a license from Invitrogen The purchase of this product conveys 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 sell or otherwise transfer a this product b its components or c materials made using this product or its components to a third party or otherwise use this product or its components or materials made using this product or its components for Commercial Purposes The buyer may transfer information or materials made through the use of this product to
9. Limited Use Label License No 51 Blasticidin and the Blasticidin Selection Marker Limited Use Label License No 108 Lentiviral Technology 44 Blasticidin and the blasticidin resistance gene bsd are the subject of U S Patent No 5 527 701 sold under patent license for research purposes only For information on purchasing a license to this product for purposes other than research contact Licensing Department Invitrogen Corporation 1600 Faraday Avenue Carlsbad California 92008 Phone 760 603 7200 Fax 760 602 6500 TM The Lentiviral Technology based upon the lentikat system is exclusively licensed from Cell Genesys Inc under U S Patent Nos 5 686 279 5 834 256 5 858 740 5 994 136 6 013 516 6 051 427 6 165 782 and 6 218 187 and corresponding patents and applications in other countries for internal research purposes only Use of this technology for gene therapy applications or bioprocessing other than for non human research use requires a license from Cell Genesys Cell Genesys Inc 342 Lakeside Drive Foster City California 94404 The purchase of this product conveys 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 including non gene therapy research and target validation applications in laboratory animals whether the buyer is an academic or for profit entity The buyer cannot sell or otherwise transfer
10. Lipofectamine 2000 handled incorrectly e Store at 4 C Do not freeze Mix gently by inversion Do not vortex Using fluorescence microscopy to view EmGFP titer The signal level of EmGFP in the cells is not optimal for visual evaluation using fluorescence microscopy We recommend using only flow cytometry to evaluate transduction efficiency No colonies obtained upon titering Too much antibiotic used for selection Determine the antibiotic sensitivity of your cell line by performing a kill curve experiment and use the minimum concentration required to kill your untransduced cell line Viral stocks stored incorrectly Aliquot and store stocks at 80 C Do not freeze thaw more than 3 times Polybrene not included during transduction Transduce the lentiviral construct into cells in the presence of Polybrene Titer indeterminable cells confluent Too little antibiotic used for selection Increase amount of antibiotic Viral supernatant insufficiently diluted Titer lentivirus using a wider range of 10 fold serial dilutions e g 10 to 10 Transducing Mammalian Cells The table below lists some potential problems and possible solutions that may help you troubleshoot your transduction and expression experiment Problem Reason Solution No expression of the gene of interest Promoter silencing e Lentiviral constructs may integrate into a chrom
11. Optional Millex HV 0 45 um PVDF filters Millipore cat no SLHVR25LS or equivalent to filter viral supernatants e Optional pLenti control vector containing EmGFP sold separately page viii Materials Supplied with Kit e ViraPower Packaging Mix resuspend in 195 ul of sterile water to a concentration of 1 ug ul e pLenti control vector containing lacZ resuspended in sterile water to a concentration of 1 ug ul TM e Lipofectamine 2000 transfection reagent mix gently before use Continued on next page 12 Producing Lentivirus in 293FT Cells Continued Forward If you are a first time user follow the procedure below to cotransfect 293FT cells Transfection For information on positive controls see page 8 We recommend including a Procedure negative control no DNA no Lipofectamine 2000 in your experiment to help you evaluate your results 1 The day before transfection Day 1 plate 293FT cells in a 10 cm tissue culture plate so that they will be 90 95 confluent on the day of transfection i e 5 x 10 cells in 10 ml of growth medium containing serum Do not include antibiotics in the medium Incubate cells overnight at 37 C in a humidified 5 CO incubator 2 On the day of transfection Day 2 remove and discard the culture medium from the 293FT cells and replace with 5 ml of growth medium Opti MEM I Medium page viii containing serum Do not use antibiotics in the medium 3 For each transfecti
12. against the buyer of infringement of the above patents based upon the manufacture use or sale of a therapeutic clinical diagnostic vaccine or prophylactic product developed in research by the buyer in which this product or its components was employed provided that none of i this product ii any of its components or iii a method claim of the foregoing patents was used in the manufacture of such product Invitrogen Corporation will not assert a claim against the buyer of infringement of the above patents based upon the use of this product to manufacture a protein for sale provided that no method claim in the above patents was used in the manufacture of such protein If the purchaser is not willing to accept the limitations of this limited use statement Invitrogen is willing to accept return of the product with a full refund For information on purchasing a license to use this product for purposes other than those permitted above contact Licensing Department Invitrogen Corporation 1600 Faraday Avenue Carlsbad California 92008 Phone 760 603 7200 For additional information about Invitrogen s policy for the use and distribution of Gateway clones see the section entitled Gateway Clone Distribution Policy page 47 Continued on next page Purchaser Notification Continued Limited Use Label License No 27 Lipofectamine 2000 Limited Use Label License No 28 CMV Promoter The purchase of this product conveys t
13. be distributed for scientific research by academic and government organizations without royalty payment to Invitrogen Organizations other than academia and government may also distribute such Gateway expression clones for a nominal fee 10 per clone payable to Invitrogen We would ask that such distributors of Gateway entry and expression clones indicate that such clones may be used only for research purposes that such clones incorporate the Gateway Technology and that the purchase of Gateway Clonase from Invitrogen is required for carrying out the Gateway recombinational cloning reaction This should allow researchers to readily identify Gateway containing clones and facilitate their use of this powerful technology in their research Use of Invitrogen s Gateway Technology including Gateway clones for purposes other than scientific research may require a license and questions concerning such commercial use should be directed to Invitrogen s licensing department at 760 603 7200 47 References Andersson S Davis D L Dahlback H J rnvall H and Russell D W 1989 Cloning Structure and Expression of the Mitochondrial Cytochrome P 450 Sterol 26 Hydroxylase a Bile Acid Biosynthetic Enzyme J Biol Chem 264 8222 8229 Boshart M Weber F Jahn G Dorsch H sler K Fleckenstein B and Schaffner W 1985 A Very Strong Enhancer is Located Upstream of an Immediate Early Gene of Human Cytomegalovi
14. lentiviral construct has integrated into the genome you may assay for transient expression of your recombinant protein or use antibiotic selection to generate a stable cell line for long term expression studies TM TM This manual provides an overview of the ViraPower HiPerform Lentiviral Expression System and provides instructions and guidelines to 1 Co transfect the pLenti based expression vector and the ViraPower Packaging Mix into the 293FT Cell Line to produce a lentiviral stock Titer the lentiviral stock Use the lentiviral stock to transduce your mammalian cell line of choice Assay for transient expression of your recombinant protein or Gom SO IS Generate a stably transduced cell line if desired For details and instructions to generate your expression vector refer to the manual for the pLenti vector you are using For instructions to culture and maintain the 293FT producer cell line refer to the 293FT Cell Line manual These manuals are supplied with the ViraPower HiPerform Lentiviral Expression Kits and are also available for downloading from www invitrogen com or by contacting Technical Support page 40 Biosafety Features of the System Introduction Biosafety Features of the ViraPower HiPerform Lentiviral System The ViraPower HiPerform Lentiviral Expression System is a third generation system based on lentiviral vectors developed by Dull et al 1998 This fourth generatio
15. maintenance in E coli Ampicillin bla resistance gene Allows selection of the plasmid in E coli 35 Map and Features of pLP2 pLP2 Map The figure below shows the features of the pLP2 vector The complete sequence of pLP2 is available for downloading from www invitrogen com or by contacting Technical Support see page 40 Comments for pLP2 4180 nucleotides RSV enhancer promoter bases 1 271 TATA box bases 200 207 Transcription initiation site base 229 RSV UTR bases 230 271 HIV 1 Rev ORF bases 391 741 HIV 1 LTR polyadenylation signal bases 850 971 bla promoter bases 1916 2014 Ampicillin bla resistance gene bases 2015 2875 pUC origin bases 3020 3693 Continued on next page 36 Map and Features of pLP2 Continued Features of pLP2 4180 bp contains the following elements Features have been functionally pLP2 tested Feature Benefit RSV enhancer promoter Permits high level expression of the rev gene Gorman et al 1982 HIV 1 Rev ORF Encodes the Rev protein that interacts with the RRE on pLP1 to induce Gag and Pol expression and on the pLenti6 V5 expression vector to promote the nuclear export of the unspliced viral RNA for packaging into viral particles HIV 1 LTR polyadenylation signal Allows efficient transcription termination and polyadenylation of mRNA Ampicillin bla resistance gene Allows selection of the plasmid in E coli
16. of 46 x 10 and 5 x 10 It is important to note that user experience the nature of the gene and vector backbone may affect virus titer If the titer of your unconcentrated virus is suitable i e 1 x 10 TU ml or higher proceed to Transduction of Cells With Lentivirus If the titer of your concentrated lentiviral stock is less than 1 x 10 TU ml we recommend producing a new lentiviral stock See Troubleshooting page 30 for more tips and guidelines to optimize your viral yield 25 Transduction and Analysis Introduction Important Transient vs Stable Expression Multiplicity of Infection MOI Determining the Optimal MOI 26 Once you have generated a lentiviral stock with a suitable titer you are ready to transduce the lentiviral construct into the mammalian cell line of choice and assay for expression of your recombinant protein Guidelines are provided below Your lentiviral construct contains a deletion in the 3 LTR that leads to self inactivation of the lentivirus after transduction into mammalian cells Once integrated into the genome the lentivirus can no longer produce packageable virus After transducing your lentiviral construct into the mammalian cell line of choice you may assay for expression of your gene of interest in the following Ways e For pLenti6 3 and pLenti 7 3 vectors pool a heterogeneous population of cells and test for expression directly after transduction i e t
17. titer 4 Add the viral supernatant to your mammalian cell line of interest Select for stably transduced cells if desired Your Mammalian Cell Line of Interest promoter gene of interest V5 l 5 Assay for recombinant protein of interest Methods General Information Introduction Positive Control Lipofectamine 2000 Opti MEM TM TM The ViraPower HiPerform Lentiviral Expression System is designed to help you create a lentivirus to deliver and express a gene of interest in mammalian cells Although the system has been designed to help you express your recombinant protein of interest in the simplest most direct fashion use of the system is geared towards those users who are familiar with the principles of retrovirus biology and retroviral vectors We highly recommend that users possess a working knowledge of virus production and tissue culture techniques For more information about these topics refer to the following published reviews e Retrovirus biology and the retroviral replication cycle see Buchschacher and Wong Staal 2000 and Luciw 1996 e Retroviral and lentiviral vectors see Naldini 1999 Naldini 1998 Yee 1999 and Pandya et al 2001 We recommend including a positive control vector in your cotransfection experiment to generate a control lentiviral stock that may be used to help you optimize expression conditions in your mammalian cell line of interest e Ea
18. you troubleshoot your cotransfection and titering experiments Problem Reason Solution Low viral titer Low transfection efficiency e Used poor quality expression construct plasmid DNA i e plasmid DNA from a mini prep e Unhealthy 293FT cells cells exhibit low viability e Cells transfected in media containing antibiotics i e Geneticin e Plasmid DNA transfection reagent ratio incorrect e Insufficient co transfection e 293FT cells plated too sparsely e Do not use mini prep plasmid DNA for transfection Use the PureLink HiPure Plasmid Midiprep kti or CsCl gradient centrifugation to prepare plasmid DNA e Use healthy 293FT cells under passage 16 do not overgrow e Although Geneticin is required for stable maintenance of 293FT cells Do not add Geneticin to media during transfection as this reduces transfection efficiency and causes cell death e Usea DNA in pg Lipofectamine 2000 in pl ratio ranging from 1 2 to 1 3 e Use more DNA Lipofectamine 2000 keeping the ratios the same For example use 5 ug of lentiviral vector 15 ug of packaging mix and 60 ul of TM Lipofectamine 2000 for transfection e Plate cells such that they are 90 95 confluent at the time of transfection or use the Reverse Transfection protocol i e add cells to media containing DNA lipid complexes see page 14 Transfected cells not cultured in media containing sodium pyruvate One da
19. 0 1320 HIV 1 gag pol sequences bases 1355 5661 gag coding sequence bases 1355 2857 gag pol frameshift base 2650 pol coding sequence bases 2650 5661 HIV 1 Rev response element RRE bases 5686 5919 Human f globin polyadenylation signal bases 6072 6837 pUC origin bases 6995 7668 C Ampicillin bla resistance gene bases 7813 8673 C bla promoter bases 8674 8772 C C complementary strand Continued on next page Map and Features of pLP1 Continued Features of pLP1 8889 bp contains the following elements Features have been functionally pLP1 tested Feature Benefit Human cytomegalovirus CMV promoter Permits high level expression of the HIV 1 gag and pol genes in mammalian cells Andersson et al 1989 Boshart et al 1985 Nelson et al 1987 Human globin intron Enhances expression of the gag and pol genes in mammalian cells HIV 1 gag coding sequence Encodes the viral core proteins required for forming the structure of the lentivirus Luciw 1996 HIV 1 pol coding sequence Encodes the viral replication enzymes required for replication and integration of the lentivirus Luciw 1996 HIV 1 Rev response element RRE Permits Rev dependent expression of the gag and pol genes Human globin polyadenylation signal Allows efficient transcription termination and polyadenylation of mRNA pUC origin of replication ori Permits high copy replication and
20. 02 6500 Email outlicensing invitrogen com Continued on next page 41 Purchaser Notification Continued Limited Use Label License No 19 Gateway Cloning Products Gateway Clone Distribution Policy 42 This product and its use is the subject of one or more of U S Patent Nos 5 888 732 6 143 557 6 171 861 6 270 969 and 6 277 608 and or other pending U S and foreign patent applications owned by Invitrogen Corporation The purchase of this product conveys 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 purchase of this product does not convey a license under any method claims in the foregoing patents or patent applications or to use this product with any recombination sites other than those purchased from Invitrogen Corporation or its authorized distributor The right to use methods claimed in the foregoing patents or patent applications with this product for research purposes only can only be acquired by the use of Clonase purchased from Invitrogen Corporation or its authorized distributors The buyer cannot modify the recombination sequence s contained in this product for any purpose The buyer cannot sell or otherwise transfer a this product b its components or c materials made by the employment of this product or its components to a third party or ot
21. 40 jpinfo invitrogen com MSDSs Material Safety Data Sheets are available on our web site at www invitrogen com msds Product qualification is described in the Certificate of Analysis CofA available on our website by product lot number at www invitrogen com cofa Invitrogen is committed to providing our customers with high quality goods and services Our goal is to ensure that every customer is 100 satisfied with our products and our service If you should have any questions or concerns about an Invitrogen product or service contact our Technical Support Representatives Invitrogen warrants that all of its products will perform according to specifications stated on the certificate of analysis The company will replace free of charge any product that does not meet those specifications This warranty limits Invitrogen Corporation s liability only to the cost of the product No warranty is granted for products beyond their listed expiration date No warranty is applicable unless all product components are stored in accordance with instructions Invitrogen reserves the right to select the method s used to analyze a product unless Invitrogen agrees to a specified method in writing prior to acceptance of the order Invitrogen makes every effort to ensure the accuracy of its publications but realizes that the occasional typographical or other error is inevitable Therefore Invitrogen makes no warranty of any kind regar
22. HiPerform Lentiviral Expression Kit also includes a pLenti Vectors vi based expression vector kit The expression vector kit includes e ApLenti based expression vector for cloning your gene of interest e A corresponding expression control plasmid e OneShot StbI3 Chemically Competent E coli for transformation Expression vectors offered in the kits include pLenti6 3 V5 TOPO vector Catalog no K5310 00 pLenti7 3 V5 TOPO vector Catalog no K5320 00 pLenti6 3 V5 DEST Gateway vector Catalog no K5330 00 and pLenti7 3 V5 DEST Gateway vector Catalog no K5340 00 Refer to the appropriate vector manual supplied with the kit for a detailed description of the reagents provided with each vector kit and instructions to generate an expression clone containing your gene of interest Continued on next page Kit Contents and Storage Continued ViraPower Lentiviral Support Kit Contents ViraPower Packaging Mix One Shot StbI3 Chemically Competent E coli Genotype of StbI3 Cells 293FT Cell Line The ViraPower HiPerform Lentiviral Support Kit includes the following vectors and reagents Store as directed below Important Store Lipofectamine 2000 at 4 C DO NOT FREEZE Reagent Composition Quantity Storage ViraPower Contains a mixture of the pLP1 195 ug 20 C Packaging Mix pLP2 and pLP VSVG plasmids lyophilized in TE pH 8 0 Lipofectamine 2000 Proprietary 0 75 ml 4
23. Invitrogen ViraPower HiPerform Lentiviral Expression Systems Lentiviral systems for high level expression in dividing and non dividing mammalian cells Catalog nos K5310 00 K5320 00 K5330 00 K5340 00 Version A 13 November 2007 A10290 ii Table of Contents Kit Contents nd Storage x adeo epe e ene audient eet det hi v Accessory Products ste ete seen te e E R eret en e e e He DH RH acne thee viii Introduction m 1 OVeEVIeWos seite trie bae tetti bare E TE tdt uccidere lares 1 Biosafety Features of the Syster ine e E e o ee RE nennt te tenente tenete eaei 5 Biosafety Features of the System Continued sse tenete tenente 6 Experimental Outline e leat eR n pa aei hel eie eta tu e te re e 7 Methods me 8 General Information gestiegen en mi dp dd di e d ren RE A s 8 Generating Your pLenti Expression Construct sse nnne tenete tenete 9 Producing Lentivirus in 293FT Cells iie a aa ghe e ovii aia eglise S 10 Titering Your Lentiviral Stock eee esq erue eiie eee ere ee made Loe 16 Titering Your Lentiviral Stock Using EmGEPP sss 18 Titering Your Lentiviral Stock Using Blasticidin neeeeenneenneenneneennnn 22 Transduction and Analysis tenete i eE E aE ENEE tenente nennen 26 TroubleshOOHng cinese Iratus ure lia E ibm plan san 30 elo RD M 33 Blasted Meerane e E a ee uo tem E et dashe
24. Kits listed above Catalog no Components K5310 00 K5320 00 K5330 00 K5340 00 pLenti6 3 V5 TOPO TA Cloning Kit Y pLenti7 3 V5 TOPO TA Cloning Kit Y pLenti6 3 V5 DEST Gateway Vector Kit Y pLenti7 3 V5 DEST Gateway Vector Kit Y ViraPower Lentiviral Support Kit Y Y Y Y One Shot Stbl3 Chemically Competent E coli Y Y Y Y 293FT Cell Line Y Y Y Y Blasticidin Y Y Continued on next page Kit Contents and Storage Continued Shipping Storage The ViraPower HiPerform Lentiviral products are shipped as described below Upon receipt store each component as detailed below Item Shipping Storage 293FT Cell Line Dry ice Liquid nitrogen Blasticidin Room temperature 20 C ViraPower Packaging Mix Room temperature 20 C ViraPower Lentiviral Support Kit Blue ice e ViraPower Packaging Mix 20 C e Lipofectamine 2000 4 C do not freeze pLenti6 3 V5 TOPO TA Cloning Kit Dry ice e Vectors 20 C e One Shot Stb13 Chemically Competent E coli 80 C pLenti7 3 V5 TOPO TA Cloning Kit Dry ice e Vectors 20 C One Shot Stb13 Chemically Competent E coli 80 C pLenti6 3 V5 DEST Gateway Vector Kit Dry ice e Vectors 20 C e One Shot Stbl3 Chemically Competent E coli 80 C pLenti7 3 V5 DEST Gateway Vector Kit Dry ice e Vectors 20 C e One Shot Stbl3 Chemically Competent E coli 80 C Expression Each ViraPower
25. The ViraPower HiPerform Lentiviral Expression Systems facilitate highly efficient in vitro delivery of a target gene to dividing and non dividing mammalian cells using a replication incompetent lentivirus Based on the lentikat system developed by Cell Genesys Dull et al 1998 the ViraPower HiPerform Lentiviral Expression System possesses features which enhance its biosafety while allowing high level gene expression in a wider range of cell types than traditional retroviral systems The System includes the following major components e ApLenti based expression vector into which the gene of interest will be cloned The vector contains the WPRE and cPPT elements for higher levels of gene expression with more cells expressing your gene of interest and faster titering times The vector also contains the elements required to allow packaging of the expression construct into virions e g 5 and 3 LTRs Y packaging signal For more information about the pLenti expression vectors refer to the manual for the specific vector you are using TM e The ViraPower Packaging Mix that contains an optimized mixture of the three packaging plasmids pLP1 pLP2 and pLP VSVG These plasmids supply the helper functions as well as structural and replication proteins in trans required to produce the lentivirus For more information about the packaging plasmids see the Appendix pages 34 39 e VSV Envelope Glycoprotein Most retroviral vectors
26. a Ci7H2NsOs HCl Sy ps N HOOC O NSA NH NH2 O Always wear gloves mask goggles and a laboratory coat when handling Blasticidin Weigh out Blasticidin and prepare solutions in a hood e Blasticidin is soluble in water and acetic acid e Prepare a stock solution of 5 to 10 mg ml Blasticidin in sterile water and filter sterilize the solution e Aliquot in small volumes suitable for one time use and freeze at 20 C for long term storage or store at 4 C for short term storage e Aqueous stock solutions are stable for 1 week at 4 C and 6 8 weeks at 20 C e pH of the aqueous solution should not exceed 7 0 to prevent inactivation of Blasticidin e Do not subject stock solutions to freeze thaw cycles do not store in a frost free freezer e Upon thawing use what you need and discard the unused portion e Medium containing Blasticidin may be stored at 4 C for up to 2 weeks 33 Map and Features of pLP1 pLP1 Map 34 The figure below shows the features of the pLP1 vector Note that the gag and pol genes are initially expressed as a gag pol fusion protein which is then self cleaved by the viral protease into individual Gag and Pol polyproteins The complete sequence of pLP1 is available for downloading from www invitrogen com or by contacting Technical Support see page 40 Comments for pLP1 8889 nucleotides CMV promoter bases 1 747 TATA box bases 648 651 Human f globin intron bases 88
27. a scientific collaborator provided that such transfer is not for any Commercial Purpose and that such collaborator agrees in writing a not to transfer such materials to any third party and b to use such transferred materials and or information solely for research and not for Commercial Purposes Commercial Purposes means any activity by a party for consideration and may include but is not limited to 1 use of the product or its components in manufacturing 2 use of the product or its components to provide a service information or data 3 use of the product or its components for therapeutic diagnostic or prophylactic purposes or 4 resale of the product or its components whether or not such product or its components are resold for use in research Invitrogen Corporation will not assert a claim against the buyer of infringement of the above patents based upon the manufacture use or sale of a therapeutic clinical diagnostic vaccine or prophylactic product developed in research by the buyer in which this product or its components was employed provided that neither this product nor any of its components was used in the manufacture of such product If the purchaser is not willing to accept the limitations of this limited use statement Invitrogen is willing to accept return of the product with a full refund For information on purchasing a license to this product for purposes other than research contact Licensing Department Invitrogen Co
28. a this product b its components or c materials made using this product or its components to a third party or otherwise use this product or its components or materials made using this product or its components for Commercial Purposes The buyer may transfer information or materials made through the use of this product to a scientific collaborator provided that such transfer is not for any Commercial Purpose and that such collaborator agrees in writing a not to transfer such materials to any third party and b to use such transferred materials and or information solely for research and not for Commercial Purposes Commercial Purposes means any activity by a party for consideration and may include but is not limited to 1 use of the product or its components in manufacturing 2 use of the product or its components to provide a service information or data 3 use of the product or its components for therapeutic diagnostic or prophylactic purposes or 4 resale of the product or its components whether or not such product or its components are resold for use in research Invitrogen Corporation will not assert a claim against the buyer of infringement of the above patents based upon the manufacture use or sale of a therapeutic clinical diagnostic vaccine or prophylactic product developed in research by the buyer in which this product or its components was employed provided that neither this product nor any of its components was used in the
29. are limited in their usefulness as gene delivery vehicles by their restricted tropism and generally low titers In the ViraPower HiPerform Lentiviral Expression System this limitation has been overcome by use of the G glycoprotein gene from Vesicular Stomatitis Virus VSV G as a pseudotyping envelope thus allowing production of a high titer lentiviral vector with a significantly broadened host cell range Burns et al 1993 Emi et al 1991 Yee et al 1994 e An optimized 293FT producer cell line that stably expresses the SV40 large T antigen under the control of the human CMV promoter and facilitates optimal production of virus For more information about the 293FT Cell Line refer to the 293FT Cell Line manual TM You will cotransfect the ViraPower Packaging Mix and the pLenti vector containing your gene of interest into 293FT cells to produce a replication incompetent lentivirus which is used to transduce a mammalian cell line of interest Continued on next page Overview Continued Features of the ViraPower HiPerform Lentiviral Systems The major Features of the ViraPower TM HiPerform Lentiviral Systems include An expression plasmid containing the gene of interest under the control a CMV early promoter and elements that allow packaging of the construct into virions Polypurine Tract from HIV cPPT for increased viral titer Park et al 2001 Woodchuck Posttranscriptional Regulatory Eleme
30. ch pLenti expression vector kit includes a positive control vector for use as an expression control e g pLenti6 3 V5 TOPO lacZ or pLenti6 3 V5 GW lacZ For more information about the positive control vector supplied with each kit refer to the appropriate expression vector manual e A control lentiviral expression vector containing Emerald Green Fluorescent Protein EmGFP for fluorescent detection pLenti6 3 V5 GW EmGEP is available separately from Invitrogen page viii The Lipofectamine 2000 reagent supplied with the kit Ciccarone et al 1999 is a proprietary cationic lipid based formulation suitable for the transfection of nucleic acids into eukaryotic cells Using Lipofectamine 2000 to transfect 293FT cells offers the following advantages e Provides the highest transfection efficiency in 293FT cells e DNA Lipofectamine 2000 complexes can be added directly to cells in culture medium in the presence of serum e Removal of complexes or medium change or addition following transfection are not required although complexes can be removed after 4 6 hours without loss of activity Note Lipofectamine 2000 is available separately from Invitrogen or as part of the ViraPower HiPerform Lentiviral Support Kits see page viii TM To facilitate optimal formation of DNA Lipofectamine 2000 complexes we recommend using Opti MEM I Reduced Serum Medium available from Invitrogen see page viii Generati
31. containing virus gently by pipetting DO NOT vortex and add to the cells Add Polybrene if desired to the plate a final concentration of 6 ug ml Swirl the plate gently to mix Incubate at 37 C in a CO incubator overnight Note If you are transducing cells with undiluted viral stock and are concerned about possible toxicity or growth effects caused by overnight incubation it is possible to incubate cells for as little as 6 hours prior to changing medium The following day Day 2 remove the medium containing virus and replace with fresh complete culture medium without Blasticidin The following day Day 3 you may analyze the cells for expression of EmGFP by flow cytometry see Important page 18 You may sort the cells expressing EmGFP with flow cytometry and use these cells for assaying protein expression Note pLenti7 3 vectors do not contain an antibiotic selection marker and therefore do not generate antibiotic resistant clones Although your gene of interest is integrated into the lentiviral genome there is no antibiotic selection pressure maintaining the integrity of the expression construct As a result depending on the influence of surrounding genomic sequences your construct may change over the course of multiple passages resulting in reduction or loss of protein expression 29 Troubleshooting Generating the Lentiviral Stock The table below lists some potential problems and possible solutions that may help
32. d A 33 Map and Feat r s of PER Ta nennen nennen ire tren imr ee t eei e n gain 34 Map and Features of pLP 2 2 2 neue aa degere ei p eo RR oed 36 Map and Features of pLP VSVG cscccecesesessstssesessseseeceeenesenesesesesesnansnesesescsnanenesesescseaesceeesessneseseseseaeeceesuenenenees 38 Techical SUpport ysa ente uve eeubentetu initial decina 40 Purchaser Notification x etit tee etai etel dese et eee p ete HL erede Bats 41 Gateway Clone Distribution Policy ose tt rena Lea unio e aiii 47 References aussehen ee n t getestet pie ager eder tre ESPERE Ue tede sientes 48 iii iv Kit Contents and Storage Types of Kits This manual is supplied with the kits listed below The ViraPower HiPerform Lentiviral Expression Kits include the ViraPower HiPerform Lentiviral Support Kit an expression vector and the 293FT producer cell line The ViraPower Lentiviral Support Kits include the ViraPower Packaging Mix TM Lipofectamine 2000 and a selection agent Product Catalog no ViraPower HiPerform Lentiviral TOPO Expression Kit K5310 00 ViraPower HiPerform Lentiviral FastTiter TOPO Expression Kit K5320 00 ViraPower HiPerform Lentiviral Gateway Expression Kit K5330 00 ViraPower HiPerform Lentiviral FastTiter Gateway Expression Kit K5340 00 System The following table shows the components associated with ViraPower Components HiPerform Lentiviral Expression
33. d EmGFP lentivirus titers in the range of 5x 10 2 x 10 TU ml To obtain higher lentivirus titer you can concentrate your virus see page 15 The titer of concentrated lentivirus stocks may be up to 1x 105 TU ml 21 Titering Your Lentiviral Stock Using Blasticidin Introduction Guidelines and protocols for titering your lentiviral stock using Blasticidin page viii are provided in this section If you wish to titer your lentiviral stock using EmGFP refer to page 18 Remember that you are working with media containing infectious virus Follow the recommended Federal and institutional guidelines for working with BL 2 organisms e Perform all manipulations within a certified biosafety cabinet e Treat media containing virus with bleach e Treat used pipettes pipette tips and other tissue culture supplies with bleach and dispose of as biohazardous waste e Wear gloves a laboratory coat and safety glasses or goggles when handling viral stocks and media containing virus Experimental To determine the titer of your lentiviral stocks using Blasticidin you will Outline 1 Prepare 10 fold serial dilutions of your lentiviral stocks 2 Transduce the different dilutions of lentivirus in the presence of the polycation Polybrene into a mammalian cell line HT1080 is recommended 3 Select for stably transduced cells using Blasticidin 4 Stain and count the number of Blasticidin resistant colonies in each dilution
34. ding the contents of any publications or documentation If you discover an error in any of our publications please report it to our Technical Support Representatives Invitrogen assumes no responsibility or liability for any special incidental indirect or consequential loss or damage whatsoever The above limited warranty is sole and exclusive No other warranty is made whether expressed or implied including any warranty of merchantability or fitness for a particular purpose Purchaser Notification Introduction Limited Use Label License No 5 Invitrogen Technology Use of the ViraPower HiPerform Lentiviral Expression Kits is covered under the licenses detailed below The purchase of this product conveys 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 sell or otherwise transfer a this product b its components or c materials made using this product or its components to a third party or otherwise use this product or its components or materials made using this product or its components for Commercial Purposes The buyer may transfer information or materials made through the use of this product to a scientific collaborator provided that such transfer is not for any Commercial Purpose and that such collaborator agrees in writing a not to transf
35. duction Experimental Outline Important 18 Guidelines and protocols for titering your lentiviral stock using Emerald Green Fluorescence EmGFP are provided in this section If you wish to titer your lentiviral stock using Blasticidin refer to page 22 Remember that you are working with media containing infectious virus Follow the recommended Federal and institutional guidelines for working with BL 2 organisms e Perform all manipulations within a certified biosafety cabinet e Treat media containing virus with bleach e Treat used pipettes pipette tips and other tissue culture supplies with bleach and dispose of as biohazardous waste e Wear gloves a laboratory coat and safety glasses or goggles when handling viral stocks and media containing virus To determine the titer of your EmGFP lentiviral stocks you will 1 Prepare a 50 fold or 20 fold serial dilutions of your lentiviral stocks 2 Transduce the different dilutions of lentivirus in the presence of the polycation Polybrene page 17 3 Determine the Lentiviral titer by fluorescence detection using flow cytometry at 2 days post transduction see Important below We do not recommend the use of fluorescence microscopy for detecting EmGFP in your cells from the pLenti7 3 vectors The pLenti7 3 vectors are designed with EmGEFP in their vector backbone which allows for quick screens of transient expression in your cells and titering times of only 2 da
36. e DNA Lipofectamine 2000 complexes and replace with 10 ml complete culture medium without antibiotics 6 Incubate cells for 48 72 hours at 37 C in a humidified 5 CO incubator Minimal differences in viral yield are observed whether supernatants are collected at either 48 or 72 hours posttransfection Note Expression of the VSV G glycoprotein causes 293FT cells to fuse resulting in the appearance of large multinucleated cells known as syncytia This morphological change is normal and does not affect production of the lentivirus 7 Posttransfection Day 4 or 5 harvest virus containing supernatants by removing and placing the medium into a 15 ml sterile capped conical tube Caution Remember that you are working with infectious virus at this stage Follow recommended guidelines for working with BL 2 organisms refer to page 5 Centrifuge supernatants at 2 000 x g for 15 minutes at 4 C to pellet debris Optional Filter the viral supernatants through a Millex HV 0 45 um or equivalent PVDF filter see Note next page 10 Pipet viral supernatants into cryovials in 1 ml aliquots 11 Store viral stocks at 80 C Proceed to Titering Your Lentiviral Stock page 16 Continued on next page 14 Producing Lentivirus in 293FT Cells Continued Note Concentrating Virus Long Term Storage Scaling Up Virus Production TM TM It should be possible to use the new ViraPower HiPerform Lentiviral vector construct
37. e Enhanced protein expression up to 4 fold or greater e Generates an HIV 1 based lentivirus that effectively transduces both dividing and non dividing mammalian cells thus broadening the potential applications beyond those of traditional Moloney Leukemia Virus MoMLV based retroviral systems Naldini 1998 e Efficiently delivers the gene of interest to mammalian cells in culture or in vivo Dull et al 1998 e Provides stable long term expression of a target gene beyond that offered by traditional adenoviral based systems Dull et al 1998 Naldini et al 1996 e Produces a pseudotyped virus with a broadened host range Yee et al 1994 e Includes multiple features designed to enhance the biosafety of the system e pLenti6 3 series vectors offer significantly improved levels of expression of your gene of interest by increasing the number of cells that express the cloned gene of interest also called the Open Reading Frame ORF e pLenti7 3 series vectors offer significantly improved levels of expression of your gene of interest pLenti7 3 vectors also allow high speed and high throughput titering applications using EmGFP and reduce the titering time down to 2 days Once the lentivirus enters the target cell the viral RNA is reverse transcribed actively imported into the nucleus Lewis amp Emerman 1994 Naldini 1999 and stably integrated into the host genome Buchschacher amp Wong Staal 2000 Luciw 1996 After the
38. e for 10 minutes at room temperature 10 Remove the crystal violet stain and wash the cells with PBS Repeat wash 11 Count the blue stained colonies and determine the titer of your lentiviral stock Continued on next page 24 Titering Your Lentiviral Stock Using Blasticidin Continued What You Should See Example of Expected Results Next Steps When titering pLenti lentiviral stocks using HT1080 cells we generally obtain titers ranging from 1 5 x 10 for unconcentrated virus up to 2 x 10 for concentrated virus transducing units TU ml In this experiment a Lenti6 3 V5 GW lacZ lentiviral stock was generated using the protocol on page 13 and was concentrated by ultracentrifugation HT1080 cells were transduced with 10 fold serial dilutions of the lentiviral supernatant 10 to 10 dilutions or untransduced mock following the protocol on page 24 At 48 hours post transduction the cells were placed under Blasticidin selection 10 pg ml After 10 days of selection the cells were stained with crystal violet see plate below and colonies were counted 10 10 mock 105 10 10 In the plate above the colony counts were e Mock no colonies e 10 dilution confluent undeterminable e 10 dilution confluent undeterminable e 10 dilution confluent undeterminable e 10 dilution 46 e 10 dilution 5 Thus the titer of this concentrated lentiviral stock is 4 8 x 10 TU ml i e average
39. entiviral stock using the mammalian cell line of choice You will use at least one 6 well plate for each lentiviral stock to be titered usually one mock well plus five dilutions 1 24 hours before transduction seed cells in a 96 well format at a density of 6 000 cells per well Incubate in a 37 C CO incubator overnight On the day of transduction Day 1 thaw your lentiviral stock In a biosafety cabinet prepare a 50 fold or 20 fold serial dilution of the Lentiviral stock in DMEM growth medium supplemented with polybrene page 17 Mix each virus dilution gently by inversion DO NOT vortex Important Do NOT dilute virus in culture medium containing Blasticidin Remove the culture medium from each well of cells and replace with the diluted virus solution We recommend allocating 3 6 replicate wells per sample Swirl the plate gently to mix Incubate at 37 C in a CO incubator overnight After 24 hours incubation Day 2 remove the virus containing media from each well and discard See Caution previous page Replace with 100 ul of fresh growth medium in each well and incubate overnight in a 37 C CO incubator Important Do NOT add Blasticidin to the growth medium After 24 hours incubation Day 3 remove the growth media from each well and discard Replace with dissociation solution above in each well Allow cells to dissociate for 5 minutes at 37 C then proceed Preparing Cells for Flow Cytometry next page see Importa
40. er such materials to any third party and b to use such transferred materials and or information solely for research and not for Commercial Purposes Commercial Purposes means any activity by a party for consideration and may include but is not limited to 1 use of the product or its components in manufacturing 2 use of the product or its components to provide a service information or data 3 use of the product or its components for therapeutic diagnostic or prophylactic purposes or 4 resale of the product or its components whether or not such product or its components are resold for use in research Invitrogen Corporation will not assert a claim against the buyer of infringement of patents owned or controlled by Invitrogen Corporation which cover this product based upon the manufacture use or sale of a therapeutic clinical diagnostic vaccine or prophylactic product developed in research by the buyer in which this product or its components was employed provided that neither this product nor any of its components was used in the manufacture of such product If the purchaser is not willing to accept the limitations of this limited use statement Invitrogen is willing to accept return of the product with a full refund For information on purchasing a license to this product for purposes other than research contact Licensing Department Invitrogen Corporation 1600 Faraday Avenue Carlsbad California 92008 Phone 760 603 7200 Fax 760 6
41. ercentage of GFP positive cells fall within the range of 1 30 White et al 1999 Sastry et al 2002 This is to avoid analyzing dilution samples containing multiple integrated lentiviral genomes which may result in an underestimate of the viral titer or dilution samples containing too few transduced cells which will give inaccurate results Titer is expressed as transducing units TU ml 1 In the following example an EmGFP lentiviral stock was generated using the protocol on the previous page The stock was concentrated and the following data were generated after performing flow cytometry Lentivirus Dilution EmGFP Positive Cells 10 91 5 10 34 6 104 44 The following formula White et al 1999 Sastry et al 2002 is used to calculate the titer F x C V x D F frequency of GFP positive cells percentage obtained divided by 100 C total number of cells in the well at the time of transduction V volume of inoculum in ml D lentivirus dilution In the above example the 10 dilution is used to calculate the titer since the percentage of EmGFP positive cells falls into the desired range of 1 30 The frequency of EmGFP positive cells is 4 4 100 0 044 multiplied by 2 x 10 the number of cells in the well divided by 1 the volume of inoculum Thus the calculation is as follows 0 044 x 200 000 1 x 10 The titer for this example is 8 8 x 10 TU ml We typically obtain unconcentrate
42. ercise extra caution when creating lentivirus carrying potential harmful or toxic genes e g activated oncogenes For more information about the BL 2 guidelines and lentivirus handling refer to the document Biosafety in Microbiological and Biomedical Laboratories 4 Edition published by the Centers for Disease Control CDC This document may be downloaded at the following address http www cdc gov od ohs biosfty bmbl4 bmbl4toc htm Handle all lentiviruses in compliance with established institutional guidelines Since safety requirements for use and handling of lentiviruses may vary at individual institutions we recommend consulting the health and safety guidelines and or officers at your institution prior to use of the ViraPower TM HiPerform Lentiviral Expression System Experimental Outline Flow Chart The diagram below describes the general steps required to express your gene of interest using the ViraPower HiPerform Lentiviral Expression System Refer to the appropriate manual for each pLenti expression vector for instructions to generate your pLenti expression construct 1 Generate the pLenti expression construct containing your gene Expression of interest Construct ViraPower Packaging Mix 2 Cotransfect the 293FT producer cell line with your pLenti expression construct and the optimized packaging mix 293FT Producer Cell Line 3 Harvest viral supernatant and determine the
43. erforming expression studies in a dividing cell line or a non primary cell line If you have more than one lentiviral construct we recommend that you titer all of the lentiviral constructs using the same mammalian cell line For more information on cells for titering see Factors Affecting Viral Titer previous page Lentivirus transduction may be enhanced if cells are transduced in the presence of hexadimethrine bromide Polybrene Sigma Catalog no H9268 For best results we recommend performing transduction in the presence of Polybrene Note however that some cells are sensitive to Polybrene e g primary neurons Before performing any transduction experiments you may want to test your cell line for sensitivity to Polybrene at a range of 0 10 pg ml If your cells are sensitive to Polybrene e g exhibit toxicity or phenotypic changes do not add Polybrene during transduction In this case cells should still be successfully transduced with your lentivirus Follow the instructions below to prepare Polybrene 1 Preparea 6 mg ml stock solution in deionized sterile water 2 Filter sterilize and dispense 1 ml aliquots into sterile microcentrifuge tubes 3 The working stock may be stored at 4 C for up to 2 weeks Store at 20 C for long term storage up to 1 year Do not freeze thaw the stock solution more than 3 times as this may result in loss of activity 17 Titering Your Lentiviral Stock Using EmGFP Intro
44. es 10010 North Torrey Pines Road La Jolla CA 92037 Attn Office of Technology Management Phone 858 453 4100 extension 1275 Fax 858 546 8093 The 293FT cell line is genetically modified and carries the pUC derived plasmid pCMVSPORT6TAg neo As a condition of sale this product must be in accordance with all applicable local legislation and guidelines including EC Directive 90 219 EEC on the contained use of genetically modified organisms Gateway Clone Distribution Policy Introduction Gateway Entry Clones Gateway Expression Clones Additional Terms and Conditions The information supplied in this section is intended to provide clarity concerning Invitrogen s policy for the use and distribution of cloned nucleic acid fragments including open reading frames created using Invitrogen s commercially available Gateway Technology Invitrogen understands that Gateway entry clones containing attL1 and attL2 sites may be generated by academic and government researchers for the purpose of scientific research Invitrogen agrees that such clones may be distributed for scientific research by non profit organizations and by for profit organizations without royalty payment to Invitrogen Invitrogen also understands that Gateway expression clones containing attB1 and attB2 sites may be generated by academic and government researchers for the purpose of scientific research Invitrogen agrees that such clones may
45. herwise use this product or its components or materials made by the employment of this product or its components for Commercial Purposes The buyer may transfer information or materials made through the employment of this product to a scientific collaborator provided that such transfer is not for any Commercial Purpose and that such collaborator agrees in writing a not to transfer such materials to any third party and b to use such transferred materials and or information solely for research and not for Commercial Purposes Notwithstanding the preceding any buyer who is employed in an academic or government institution may transfer materials made with this product to a third party who has a license from Invitrogen under the patents identified above to distribute such materials Transfer of such materials and or information to collaborators does not convey rights to practice any methods claimed in the foregoing patents or patent applications Commercial Purposes means any activity by a party for consideration and may include but is not limited to 1 use of the product or its components in manufacturing 2 use of the product or its components to provide a service information or data 3 use of the product or its components for therapeutic diagnostic or prophylactic purposes or 4 resale of the product or its components whether or not such product or its components are resold for use in research Invitrogen Corporation will not assert a claim
46. in as appropriate Replenish the selective media every 3 4 days and observe the percentage of surviving cells Determine the appropriate concentration of Blasticidin that kills the cells within 10 14 days after addition of antibiotic You will need the following items Your pLenti lentiviral stock from either the pLenti6 3 V5 TOPO vector or pLenti6 3 V5 DEST Gateway vector store at 80 C until use Adherent mammalian cell line of choice Complete culture medium for your cell line 6 mg ml Polybrene optional see page 17 6 well tissue culture plates Blasticidin 10 mg ml stock as appropriate for selection supplied with kit Crystal violet Sigma Catalog no C3886 prepare a 1 crystal violet solution in 10 ethanol Phosphate Buffered Saline PBS page viii Continued on next page 23 Titering Your Lentiviral Stock Using Blasticidin Continued Transduction and Follow the procedure below to determine the titer of your lentiviral stock using Titering Procedure the mammalian cell line of choice You will use at least one 6 well plate for every Blasticidin lentiviral stock to be titered one mock well plus five dilutions 1 The day before transduction trypsinize and count the cells plating cells ina 6 well plate at a density of 2 x 10 cells per well such that they will be 30 50 confluent at the time of transduction Incubate cells at 37 C overnight in ahumidified 5 CO incubator Example When
47. ine of interest It is possible to scale up the cotransfection experiment to produce a larger volume of lentivirus if desired For example we have scaled up the cotransfection experiment from a 10 cm plate to a T 175 cm flask and harvested up to 30 ml of viral supernatant If you wish to scale up your cotransfection remember that you will need to increase the number of cells plated and the amounts of DNA Lipofectamine 2000 and medium used in proportion to the difference in surface area of the culture vessel 15 Titering Your Lentiviral Stock Introduction ViraPower HiPerform Lentiviral FastTiter Expression Kits Factors Affecting Viral Titer 16 Before proceeding to transduction and expression experiments we highly recommend determining the titer of your lentiviral stock While this procedure is not required for some applications it is necessary if e You wish to control the number of integrated copies of the lentivirus e You wish to generate reproducible expression results Guidelines and protocols are provided in this section to titer your lentiviral stock In addition to higher expression of the gene of interest all ViraPower HiPerform Lentiviral vectors yield a higher Blasticidin Bsd or Emerald Green Fluorescence EmGFP titer compared to previous pLenti vectors The pLenti6 3 vectors K5310 00 and K5330 00 contain Bsd in the vector backbone which allows titer of active virus by selec
48. ity by a party for consideration and may include but is not limited to 1 use of the product or its components in manufacturing 2 use of the product or its components to provide a service information or data 3 use of the product or its components for therapeutic diagnostic or prophylactic purposes and or 4 resale of the product or its components whether or not such product or its components are resold for use in research In addition any use of WPRE outside of this product or the product s authorized use requires a separate license from the Salk Institute Invitrogen will not assert a claim against the buyer of infringement of patents owned by Invitrogen and claiming this product based upon the manufacture use or sale of a therapeutic clinical diagnostic vaccine or prophylactic product developed in research by the buyer in which this product or its components was employed provided that neither this product nor any of its components was used in the manufacture of such product or for a Commercial Purpose If the purchaser is not willing to accept the limitations of this limited use statement Invitrogen is willing to accept return of the product with a full refund For information on purchasing a license to this product for purposes other than research contact Licensing Department Invitrogen Corporation 1600 Faraday Avenue Carlsbad California 92008 Phone 760 603 7200 Fax 760 602 6500 or The Salk Institute for Biological Studi
49. l vector manual for assay methods Viral supernatants are generated by harvesting spent media containing virus from the 293FT producer cells Spent media lacks nutrients and may contain some toxic metabolic waste products If you are using a large volume of viral supernatant to transduce your mammalian cell line e g 1 ml of viral supernatant per well in a 6 well plate note that growth characteristics or morphology of the cells may be affected during transduction These effects are generally alleviated after transduction when the media is replaced with fresh complete media You will need the following items e Your titered lentiviral stock page 16 store at 80 C until use e Mammalian cell line of choice e Complete culture medium for your cell line e 6mg ml Polybrene if desired page 17 e Appropriately sized tissue culture plates for your application e Blasticidin as appropriate if selecting for stably transduced cells pLenti6 3 vectors only Continued on next page 27 Transduction and Analysis Continued Transduction Procedure for Blasticidin Note Detecting Recombinant Protein 28 Follow the procedure below to transduce the mammalian cell line of choice using the pLenti6 3 vectors 1 Plate cells in complete media as appropriate for your application 2 On the day of transduction Day 1 thaw your lentiviral stock and if necessary dilute the appropriate amount of virus into fresh comple
50. ld increase in viral titer Both WPRE and cPPT together produce at least a four fold increase in protein expression in most cell types compared to other vectors that do not contain these elements The ViraPower HiPerform Lentiviral FastTiter Expression Systems Catalog nos K5320 00 and K5340 00 allow for an accurate titer of functional lenti virus in just two days using EmGFP The ViraPower HiPerform Lentiviral Expression System vectors also contain e Human cytomegalovirus CMV immediate early promoter to control high level expression of the gene of interest in all four vectors e C terminal V5 tag for convenient detection e SV40 promoter driving expression of Blasticidin pLenti6 3 V5 DEST Gateway vector and pLenti6 3 V5 TOPO Vector or EmGFP pLenti7 3 V5 DEST Gateway vector and pLenti7 3 V5 TOPO vector e Blasticidin Izumi et al 1991 Kimura et al 1994 Takeuchi et al 1958 Yamaguchi et al 1965 resistance gene for stable transduction and selection in E coli and mammalian cells pLenti6 3 V5 DEST Gateway and pLenti6 3 V5 TOPO TA vectors only or e Emerald Green Fluorescent Protein EmGFP derived from Aequorea Victoria GFP pLenti7 3 V5 DEST Gateway and pLenti7 3 V5 TOPO vectors only which allows you to easily determine the Lentiviral titer by flow cytometry Continued on next page Overview Continued Components of the ViraPower HiPerform Lentiviral Expression System
51. lone to assay for expression of the recombinant protein Integration of the lentivirus into the genome is random Depending upon the influence of the surrounding genomic sequences at the integration site you may see varying levels of recombinant protein expression from different antibiotic resistant clones We recommend testing at least 5 antibiotic resistant clones and selecting the clone that provides the optimal expression of your recombinant protein for further studies You may use any method of choice to detect your recombinant protein of interest including functional analysis immunofluorescence or western blot If you have cloned your gene of interest in frame with an epitope tag you may easily detect your recombinant protein in a western blot using an antibody to the epitope tag see your lentiviral vector manual for details Transduction and Analysis Continued Transduction Procedure for EmGFP Follow the procedure below to transduce the mammalian cell line of choice using the pLenti7 3 vectors 1 2 Plate cells in complete media as appropriate for your application On the day of transduction Day 1 thaw your lentiviral stock and dilute if necessary the appropriate amount of virus see Determining Optimal MOI page 26 into fresh complete medium Keep the total volume of medium containing virus as low as possible to maximize transduction efficiency Remove the culture medium from the cells Mix the medium
52. malian Cells Exp Cell Res 197 229 233 Kimura M Takatsuki A and Yamaguchi I 1994 Blasticidin S Deaminase Gene from Aspergillus terreus BSD A New Drug Resistance Gene for Transfection of Mammalian Cells Biochim Biophys ACTA 1219 653 659 Lewis P F and Emerman M 1994 Passage Through Mitosis is Required for Oncoretroviruses but not for the Human Immunodeficiency Virus J Virol 68 510 516 Luciw P A 1996 in Fields Virology Fields B N Knipe D M Howley P M Chanock R M Melnick J L Monath T P Roizman B and Straus S E eds 3rd Ed pp 1881 1975 Lippincott Raven Publishers Philadelphia PA Naldini L 1998 Lentiviruses as Gene Transfer Agents for Delivery to Non dividing Cells Curr Opin Biotechnol 9 457 463 Naldini L 1999 in The Development of Human Gene Therapy Friedmann T ed pp 47 60 Cold Spring Harbor Laboratory Press Cold Spring Harbor NY Naldini L Blomer U Gage F H Trono D and Verma I M 1996 Efficient Transfer Integration and Sustained Long Term Expression of the Transgene in Adult Rat Brains Injected with a Lentiviral Vector Proc Natl Acad Sci USA 93 11382 11388 Nelson J A Reynolds Kohler C and Smith B A 1987 Negative and Positive Regulation by a Short Segment in the 5 Flanking Region of the Human Cytomegalovirus Major Immediate Early Gene Molec Cell Biol 7 4125 4129 Pandya S Klimatcheva E and Planelles V
53. more patents or patent applications Users of these products should determine if any licenses are required Invitrogen Corporation will not assert a claim against the buyer of infringement of patents owned by Invitrogen and claiming this product based upon the manufacture use or sale of a therapeutic clinical diagnostic vaccine or prophylactic product developed in research by the buyer in which this product or its components was employed provided that neither this product nor any of its components was used in the manufacture of such product If the purchaser is not willing to accept the limitations of this limited use statement Invitrogen is willing to accept return of the product with a full refund For information on purchasing a license to this product for purposes other than research contact Licensing Department Invitrogen Corporation 1600 Faraday Avenue Carlsbad California 92008 Phone 760 603 7200 Fax 760 602 6500 The use of the CMV promoter is covered under U S Patent Nos 5 168 062 and 5 385 839 owned and licensed by the University of Iowa Research Foundation and is sold for research use only Commercial users must obtain a license to these patents directly from the University of Iowa Research Foundation UIRF 214 Technology Innovation Center Iowa City Iowa 52242 For further information please contact the Associate Director of UIRF at 319 335 4546 Continued on next page 43 Purchaser Notification Continued
54. n lentiviral system includes a significant number of safety features designed to enhance its biosafety and to minimize its relation to the wild type human HIV 1 virus These safety features are discussed below TM TM The ViraPower HiPerform Lentiviral Expression System includes the following key safety features The pLenti expression vector contains a deletion in the 3 LTR AUS that does not affect generation of the viral genome in the producer cell line but results in self inactivation of the lentivirus after transduction of the target cell Yee et al 1987 Yu et al 1986 Zufferey et al 1998 Once integrated into the transduced target cell the lentiviral genome is no longer capable of producing packageable viral genome The number of genes from HIV 1 that are used in the system has been reduced to three i e gag pol and rev The VSV G gene from Vesicular Stomatitis Virus is used in place of the HIV 1 envelope Burns et al 1993 Emi et al 1991 Yee et al 1994 Genes encoding the structural and other components required for packaging the viral genome are separated onto four plasmids All four plasmids have been engineered not to contain any regions of homology with each other to prevent undesirable recombination events that could lead to the generation of a replication competent virus Dull et al 1998 Although the three packaging plasmids allow expression in trans of proteins required to produce viral pr
55. ng Your pLenti Expression Construct Introduction DNA Isolation Guidelines Important To generate a pLenti expression construct containing your gene of interest refer to your specific vector s manual for instructions Once you have created your expression construct isolate plasmid DNA for transfection Important You should verify that your lentiviral plasmid has not undergone aberrant recombination by performing an appropriate restriction enzyme digest See the vector manual for details Plasmid DNA for transfection into eukaryotic cells must be very clean and free from contamination with phenol and sodium chloride Contaminants will kill the cells and salt will interfere with lipid complexing decreasing transfection efficiency When performing plasmid DNA isolation with commercially available kits from E coli strains such as Stbl3 that are wild type for endonuclease 1 endA1 ensure that Solution I of the Lysis or Resuspension Buffer contains 10 mM EDTA EDTA will inactivate the endonuclease and avoid DNA nicking and vector degradation Alternatively follow the instructions included the plasmid purification kits for endA1 E coli strains Do not use mini prep plasmid DNA for lentivirus production We recommend preparing lentiviral plasmid DNA using the PureLink HiPure Plasmid MidiPrep which contains 10 mM EDTA in the Resuspension Buffer page viii Producing Lentivirus in 293FT Cells Introduction Vi
56. nome is approximately 10 kb Since the size of the elements required for expression from pLenti vectors total approximately 4 4 4 kb the size of your insert should not exceed 5 6 kb e The characteristics of the cell line used for titering We strongly recommend the human fibrosarcoma line HT1080 as the gold standard for reproducibly titering lentivirus However other cell lines may be used In general these cells should be an adherent non migratory cell line and exhibit a doubling time in the range of 18 25 hours e The age of your lentiviral stock Viral titers may decrease with long term gt 1 year storage at 80 C If your lentiviral stock has been stored for longer than 6 months we recommend titering your lentiviral stock prior to use e Number of freeze thaw cycles Viral titers can decrease as much as 10 with each freeze thaw cycle e Improper storage of your lentiviral stock Lentiviral stocks should be stored at 80 C in cryovials Continued on next page Titering Your Lentiviral Stock Continued Selecting a Cell Line for Titering Using Polybrene During Transduction Preparing and Storing Polybrene We strongly recommend the human fibrosarcoma line HT1080 ATCC cat no CCL 121 as the gold standard for reproducibly titering lentivirus However you may wish to use the same mammalian cell line to titer your lentiviral stocks as you will use to perform your expression studies e g if you are p
57. nt WPRE for increased transgene expression Zufferey et al 1999 An optimized mix of the three packaging plasmids pLP1 pLP2 and pLP VSVG that supply the structural and replication proteins in trans that are required to produce the lentivirus The 293FT cell line which allows production of lentivirus following cotransfection of the expression plasmid and the plasmids in the packaging mix Control expression plasmid to optimize virus production and cell transduction containing either e The lacZ gene which when packaged into virions and transduced into a mammalian cell line expresses galactosidase included with each expression vector kit or e The Emerald Green Fluorescent Protein EmGFP gene which when packaged into virions and transduced into a mammalian cell line expresses EmGFP available separately see page viii For more information on expression vectors and the corresponding positive control vectors refer to the manual for the specific expression or control vector you are using Overview Continued Advantages of the System How Lentivirus Works Purpose of this Manual Use of the ViraPower HiPerform Lentiviral Expression System to facilitate lentiviral based expression of the gene of interest provides the following advantages e Offers you a choice to use either Gateway technology Catalog nos K5310 00 and K5330 00 or TOPO Cloning technologies Catalog nos K5320 00 K5340 00
58. nt previous page Continued on next page 19 Titering Your Lentiviral Stock Using EmGFP Continued If you wish to fix your cells before flow cytometry you can use 2 Note formaldehyde or paraformaldehyde in calcium magnesium free PBS However these fixatives may increase autofluorescence of the cells thus it is critical to include fixed mock transduced cells as a negative control for flow cytometry detection parameters Preparing Cells Prepare cells for flow cytometry using a FITC filter according to the established for Flow protocols in use at your flow cytometry facility The steps below provide simple Cytometry guidelines and other methods may be suitable 1 After cell dissociation Steps 6 7 previous page spin cells at 2 000 x g ina centrifuge to remove residual media components and resuspend the cell pellet in flow cytometry buffer such as calcium magnesium free PBS with 1 FBS at the required density for analysis on your flow cytometer Fixing the cells is not necessary but may be done see Note above 2 Use the mock transduced cells and the lowest dilution of virus i e 10 as the negative and positive samples respectively to set up the parameters of your flow cytometer Continued on next page 20 Titering Your Lentiviral Stock Using EmGFP Continued Calculating Lentiviral Titer for EmGFP What You Can Expect EmGEFP lentivirus titers should be calculated from the dilutions at which the p
59. o 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 sell or otherwise transfer a this product b its components or c materials made using this product or its components to a third party or otherwise use this product or its components or materials made using this product or its components for Commercial Purposes The buyer may transfer information or materials made through the use of this product to a scientific collaborator provided that such transfer is not for any Commercial Purpose and that such collaborator agrees in writing a to not transfer such materials to any third party and b to use such transferred materials and or information solely for research and not for Commercial Purposes Commercial Purposes means any activity by a party for consideration and may include but is not limited to 1 use of the product or its components in manufacturing 2 use of the product or its components to provide a service information or data 3 use of the product or its components for therapeutic diagnostic or prophylactic purposes or 4 resale of the product or its components whether or not such product or its components are resold for use in research Use of this product in conjunction with methods for the introduction of RNA molecules into cells may require licenses to one or
60. obin polyadenylation signal Allows efficient transcription termination and polyadenylation of mRNA pUC origin of replication ori Permits high copy replication and maintenance in E coli Ampicillin bla resistance gene Allows selection of the plasmid in E coli 39 Technical Support Web Resources Visit the Invitrogen web site at www invitrogen com for e Technical resources including manuals vector maps and sequences application notes MSDSs FAQs formulations citations handbooks etc e Complete technical support contact information e Access to the Invitrogen Online Catalog e Additional product information and special offers Contact Us For more information or technical assistance call write fax or email Additional international offices are listed on our web site www invitrogen com Corporate Headquarters Japanese Headquarters European Headquarters Invitrogen Corporation Invitrogen Japan Invitrogen Ltd 1600 Faraday Avenue LOOP X Bldg 6F Inchinnan Business Park Carlsbad CA 92008 USA 3 9 15 Kaigan 3 Fountain Drive Tel 1 760 603 7200 Minato ku Tokyo 108 0022 Paisley PA4 9RF UK Tel Toll Free 1 800 955 6288 Tel 81 3 5730 6509 Tel 44 0 141 814 6100 Fax 1 760 602 6500 Fax 81 3 5730 6519 Tech Fax 44 0 141 814 6117 E mail tech_support invitrogen com E mail E mail eurotech invitrogen com MSDS Certificate of Analysis Limited Warranty
61. oduce lentiviral stocks in 293FT cells using the following optimized transfection conditions in the table below The amount of lentivirus produced using these recommended conditions 10 ml of virus at a titer of at least 1 x 10 transducing units TU ml is generally sufficient to transduce at least 1 x 10 cells at a multiplicity of infection MOI 1 For example 10 wells of cells plated at 1 x 10 cells well in 6 well plates could each be transduced with 1 ml of a 1 x 10 TU ml virus stock to achieve an MOI of 1 Condition Quantity Tissue culture plate size 10 cm one per lentiviral construct Number of 293FT cells to transfect 6 x 10 cells see Recommendation previous page to prepare cells for transfection Amount of ViraPower Packaging Mix 9 ug 9 pl of 1 ug ul stock Amount of pLenti expression plasmid 3 ug Amount of Lipofectamine 2000 36 ul Note You may produce lentiviral stocks using other tissue culture formats but keep in mind that optimization may be necessary to obtain the expected titers If you are producing lentivirus for the first time using the ViraPower System and 293FT cells you should perform the Forward Transfection procedure on page 13 This procedure requires plating the 293FT cells the day before transfection to obtain cells that are 90 95 confluent Note In previous ViraPower manuals this protocol was referred to as the Alternate Transfection Method If yo
62. of Human Gene Therapy Friedmann T ed pp 21 45 Cold Spring Harbor Laboratory Press Cold Spring Harbor NY Yee J K Moores J C Jolly D J Wolff J A Respess J G and Friedmann T 1987 Gene Expression from Transcriptionally Disabled Retroviral Vectors Proc Natl Acad Sci USA 84 5197 5201 Yu S F Ruden T v Kantoff P W Garber C Seiberg M Ruther U Anderson W F Wagner E F and Gilboa E 1986 Self Inactivating Retroviral Vectors Designed for Transfer of Whole Genes into Mammalian Cells Proc Natl Acad Sci USA 83 3194 3198 Zufferey R Dull T Mandel R J Bukovsky A Quiroz D Naldini L and Trono D 1998 Self inactivating lentivirus vector for safe and efficient in vivo gene delivery J Virol 72 9873 9880 2007 Invitrogen Corporation All rights reserved Polybrene is a registered trademark of Abbott Laboratories For research use only Not intended for any animal or human therapeutic or diagnostic use 49 invitrogen Corporate Headquarters Invitrogen Corporation 1600 Faraday Avenue Carlsbad CA 92008 T 1 760 603 7200 F 1 760 602 6500 E tech service invitrogen com For country specific contact information visit our web site at www invitrogen com
63. ogeny e g gal pol rev env in the 293FT producer cell line none of them contain LTRs or the Y packaging sequence This means that none of the HIV 1 structural genes are actually present in the packaged viral genome and thus are never expressed in the transduced target cell No new replication competent virus can be produced The lentiviral particles produced in this system are replication incompetent and only carry the gene of interest No other viral species are produced Expression of the gag and pol genes from pLP1 has been rendered Rev dependent by virtue of the HIV 1 RRE in the gag pol mRNA transcript Addition of the RRE prevents gag and pol expression in the absence of Rev Dull et al 1998 A constitutive promoter RSV promoter has been placed upstream of the 5 LTR in the pLenti expression vector to offset the requirement for Tat in the efficient production of viral RNA Dull et al 1998 Continued on next page Biosafety Features of the System Continued Biosafety Level 2 Important Despite the inclusion of the safety features discussed on the previous page the lentivirus produced with this System can still pose some biohazardous risk since it can transduce primary human cells For this reason we highly recommend that you treat lentiviral stocks generated using this System as Biosafety Level 2 BL 2 organisms and strictly follow all published BL 2 guidelines with proper waste decontamination Furthermore ex
64. on sample prepare DNA Lipofectamine 2000 complexes as follows a Ina sterile 5 ml tube dilute 9 ug of the ViraPower Packaging Mix and 3 ug of your pLenti expression plasmid DNA 12 ug total in 1 5 ml of Opti MEM I Medium without serum Mix gently b Ina separate sterile 5 ml tube dilute 36 ul Lipofectamine 2000 mix gently before use in 1 5 ml of Opti MEM I Medium without serum Mix gently and incubate for 5 minutes at room temperature c After incubation combine the diluted DNA Step a with the diluted Lipofectamine 2000 Step b Mix gently d Incubate for 20 minutes at room temperature to allow the DNA Lipofectamine 2000 complexes to form The solution may appear cloudy but this will not impede the transfection 4 Add all the DNA Lipofectamine 2000 complexes dropwise to the plate of 293FT cells Steps 1 and 2 Mix gently by rocking the plate back and forth Incubate the cells overnight at 37 C in a humidified 5 CO incubator 5 The next day Day 3 remove the cell culture plate containing the 293FT cells with DNA Lipofectamine complexes from the incubator Remove and discard the medium containing the DNA Lipofectamine 2000 complexes and replace with 10 ml complete culture medium without antibiotics 6 Incubate cells for 48 72 hours at 37 C in a humidified 5 CO incubator Minimal differences in viral yield are observed whether supernatants are collected at either 48 or 72 hours post transfection
65. onstructs containing activated oncogenes or potentially harmful genes is not recommended Cytotoxic effects Large volume of viral observed after supernatant used for transduction transduction e Remove the spent media containing virus and replace with fresh complete media e Concentrate the virus Yee 1999 Polybrene used during transduction Verify the sensitivity of your cells to Polybrene If cells are sensitive omit the Polybrene during transduction Too much antibiotic used for selection Determine the antibiotic sensitivity of your cell line by performing a kill curve Use the minimum concentration of antibiotic required to kill your untransduced cell line Gene of interest is toxic to cells Try a different cell line 32 Blasticidin Description Molecular Weight Formula and Structure Handling Blasticidin Preparing and Storing Stock Solutions Appendix Blasticidin S HCl is a nucleoside antibiotic isolated from Streptomyces griseo chromogenes which inhibits protein synthesis in both prokaryotic and eukaryotic cells Resistance is conferred by expression of either one of two Blasticidin S deaminase genes BSD from Aspergillus terreus Kimura et al 1994 or bsr from Bacillus cereus Izumi et al 1991 These deaminases convert Blasticidin S to a non toxic deaminohydroxy derivative Izumi et al 1991 Merck Index 12 1350 NH2 MW 458 9 Formul
66. osomal region that silences the CMV promoter Screen multiple antibiotic resistant clones and select the one with the highest expression levels Viral stocks stored incorrectly Aliquot and store stocks at 80 C Do not freeze thaw more than 3 times Frozen cells used for expression experiments for pLenti7 3 vector pLenti7 3 vectors are designed for transient expression We do not recommend using frozen cells for these expression experiments Continued on next page 31 Troubleshooting Continued Transducing Mammalian Cells continued Problem Reason Solution Poor expression of the Low transduction efficiency gene of interest e Polybrene not included during transduction e Non dividing cell type used e Transduce the lentiviral construct into cells in the presence of Polybrene e Transduce your lentiviral construct into cells using a higher MOI MOI too low Transduce your lentiviral construct into cells using a higher MOI Too much antibiotic used for selection Determine the antibiotic sensitivity of your cell line by performing a kill curve Use the minimum antibiotic concentration required to kill your untransduced cell line Cells harvested too soon after transduction Do not harvest cells until at least 48 72 hours after transduction to allow expressed protein to accumulate in transduced cells Gene of interest is toxic to cells Generating c
67. raPower Packaging Mix 293FT Cell Line SANEND 7 7 Y o P E 10 Before you can create a stably transduced cell line expressing your gene of interest you will first need to produce a lentiviral stock containing the packaged pLenti expression construct by cotransfecting the optimized packaging plasmid mix and your pLenti expression construct into the 293FT Cell Line This section provides protocols and instructions to generate a lentiviral stock The pLP1 pLP2 pLP VSVG plasmids are provided in an optimized mixture to facilitate viral packaging of your pLenti expression vector following cotransfection into 293FT producer cells The amount of the packaging mix 195 ug and Lipofectamine 2000 transfection reagent 0 75 ml supplied in the ViraPower Lentiviral Expression kit is sufficient to perform 20 cotransfections in 10 cm plates To use the ViraPower Packaging Mix resuspend the contents of one tube 195 ug in 195 ul of sterile water to obtain a 1 g pl stock Note ViraPower Packaging Mix is available separately from Invitrogen or as part of the TM ViraPower Lentiviral Support Kits page viii The human 293FT Cell Line is supplied with the ViraPower HiPerform Lentiviral Expression kits to facilitate optimal lentivirus production Naldini et al 1996 The 293FT Cell Line a derivative of the 293F Cell Line stably and constitutively expresses the SV40 large T antigen from pCMVSPORT6TA g neo and m
68. ransient expression Note that you must wait for a minimum of 48 72 hours after transduction before harvesting your cells to allow expressed protein to accumulate in transduced cells e For pLenti63 vectors only select for stably transduced cells using Blasticidin This requires a minimum of 10 12 days after transduction but allows generation of clonal cell lines that stably express the gene of interest Please be aware that the pLenti7 3 vectors are used for transient expression only and do not produce stably transduced cells Note We have observed stable expression of a target gene for at least 6 weeks following transduction and selection To obtain optimal expression of your gene of interest you will need to transduce the lentiviral construct into your mammalian cell line of choice using a suitable MOI MOI is defined as the number of virus particles per cell and generally correlates with the number of integration events and as a result expression of your gene of interest Typically expression levels increase linearly as the MOI increases A number of factors can influence optimal MOI including the nature of your mammalian cell line e g non dividing vs dividing cell type see Recommendation on the next page its transduction efficiency your application of interest and the nature of your gene of interest If you are transducing your lentiviral construct into the mammalian cell line of choice for the first time we recommend
69. rporation 1600 Faraday Avenue Carlsbad California 92008 Phone 760 603 7200 Fax 760 602 6500 Continued on next page 45 Purchaser Notification Continued Limited Use Label License No 308 WPRE Element in Lentiviral Vectors Information for European Customers 46 This product contains the Woodchuck Post transcriptional Regulatory Element WPRE which is the subject of intellectual property owned by The Salk Institute for Biological Studies and licensed to Invitrogen Corporation The purchase of this product conveys 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 sell or otherwise transfer a this product b its components or c materials made using this product or its components to a third party or otherwise use this product or its components or materials made using this product or its components for Commercial Purposes The buyer may transfer information or materials made through the use of this product to a scientific collaborator provided that such transfer is not for any Commercial Purpose and that such collaborator agrees in writing a not to transfer such materials to any third party and b to use such transferred materials and or information solely for research and not for Commercial Purposes Commercial Purposes means any activ
70. rus Cell 41 521 530 Buchschacher G L Jr and Wong Staal F 2000 Development of Lentiviral Vectors for Gene Therapy for Human Diseases Blood 95 2499 2504 Burns J C Friedmann T Driever W Burrascano M and Yee J K 1993 Vesicular Stomatitis Virus G Glycoprotein Pseudotyped Retroviral Vectors Concentration to a Very High Titer and Efficient Gene Transfer into Mammalian and Nonmammalian Cells Proc Natl Acad Sci USA 90 8033 8037 Ciccarone V Chu Y Schifferli K Pichet J P Hawley Nelson P Evans K Roy L and Bennett S 1999 Lipofectamine 2000 Reagent for Rapid Efficient Transfection of Eukaryotic Cells Focus 21 54 55 Dull T Zufferey R Kelly M Mandel R J Nguyen M Trono D and Naldini L 1998 A Third Generation Lentivirus Vector with a Conditional Packaging System J Virol 72 8463 8471 Emi N Friedmann T and Yee J K 1991 Pseudotype Formation of Murine Leukemia Virus with the G Protein of Vesicular Stomatitis Virus J Virol 65 1202 1207 Gorman C M Merlino G T Willingham M C Pastan I and Howard B H 1982 The Rous Sarcoma Virus Long Terminal Repeat is a Strong Promoter When Introduced into a Variety of Eukaryotic Cells by DNA mediated Transfection Proc Natl Acad Sci USA 79 6777 6781 Izumi M Miyazawa H Kamakura T Yamaguchi I Endo T and Hanaoka F 1991 Blasticidin S Resistance Gene bsr A Novel Selectable Marker for Mam
71. s for in vivo applications however we have not yet tested the new constructs in vivo If you plan to use your lentiviral construct for in vivo applications we recommend filtering your viral supernatant through a sterile 0 45 um low protein binding filter after the low speed centrifugation step Step 8 previous page to remove any remaining cellular debris We recommend using Millex HV 0 45 um PVDF filters Millipore Catalog no SLHVR25LS for filtration If you wish to concentrate your viral stock to obtain a higher titer perform the filtration step first before concentrating your viral stock It is possible to concentrate VSV G pseudotyped lentiviruses using a variety of methods without significantly affecting their ability to transduce cells If your cell transduction experiment requires that you use a relatively high Multiplicity of Infection MOI you may wish to concentrate your virus before titering and proceeding to transduction For details and guidelines to concentrate your virus supernatant by ultracentrifugation refer to published reference sources Yee 1999 Store viral stocks at 80 C in cryovials for long term storage Repeated freezing and thawing is not recommended as it may result in loss of viral titer When stored properly viral stocks of an appropriate titer should be suitable for use for up to one year After long term storage we recommend retitering your viral stocks before transducing your mammalian cell l
72. te medium to obtain a suitable MOI Keep the total volume of medium containing virus as low as possible to maximize transduction efficiency Do not vortex 3 Remove the culture medium from the cells Mix the medium containing virus gently by pipetting and add to the cells 4 Add Polybrene if desired to a final concentration up to 10 pg ml Swirl the plate gently to mix Incubate at 37 C in a humidified 5 CO incubator overnight Note If you are transducing cells with undiluted viral stock and are concerned about possible toxicity or growth effects caused by overnight incubation it is possible to incubate cells for as little as 6 hours prior to changing medium 5 The following day Day 2 remove the medium containing virus and replace with fresh complete culture medium Incubate at 37 C in a humidified 5 CO incubator overnight 6 The following day Day 3 perform one of the following e Harvest the cells and assay for expression of your recombinant protein if you are performing transient expression experiments e Remove the medium and replace with fresh complete medium containing the appropriate amount of Blasticidin to select for stably transduced cells Proceed to Step 7 7 Replace medium with fresh medium containing antibiotic every 3 4 days until antibiotic resistant colonies can be identified generally 10 12 days after selection 8 Pick at least 5 antibiotic resistant colonies see Note below and expand each c
73. tion of Blasticidin resistant clones after transduction Alternatively pLenti7 3 FastTiter vectors K5320 00 and K5340 00 contain the EmGFP reporter gene in the vector backbone which allows titer by flow cytometry in only 2 days post transduction For Titering lentiviral stock using EmGFP refer to page 18 For Titering lentiviral stock using Blasticidin refer to page 22 TM TM TM ViraPower HiPerform Lentiviral FastTiter Expression kits K5320 00 and K5340 00 allow you to titer lentivirus in only 2 days because the pLenti7 3 vectors contain EmGFP reporter gene in the vector backbone instead of Bsd This feature makes these kits ideal for high throughput and quick screens of transient expression using flow cytometry Important The FastTiter Expression kits are optimal for quick screens of transient expressions using flow cytometry The signal intensity produced by these kits is not optimal for detection using fluorescence microscopy We recommend flow cytometry to detect the EmGFP in your transduced cells A number of factors can influence viral titers including e The size of your gene of interest Titers will decrease as the size of the insert increases We have determined that virus titer drops approximately 2 fold for each kb over 4 kb of insert size If you wish to produce lentivirus with an insert of 4 kb you will need to concentrate the virus to obtain a suitable titer see page 15 The size of the wild type HIV ge
74. u are an experienced lentivirus user and are familiar with the growth characteristics of 293FT cells you may choose to perform the Reverse Transfection procedure on page 14 In this procedure 293FT cells are added to media containing the DNA Lipofectamine 2000 complexes Continued on next page 11 Producing Lentivirus in 293FT Cells Continued Materials Needed You willneed the following items e pLenti expression vector containing your gene of interest 0 1 3 0 ug ul in sterile water or TE pH 8 0 e 293FT cells cultured in the appropriate medium i e D MEM containing 10 FBS 4 mM L Glutamine 1 mM MEM sodium pyruvate 0 1 mM MEM Non Essential Amino Acids and 1 penicillin streptomycin and 500 pg ml Geneticin Note MEM Sodium Pyruvate provides an extra energy source for the cells and is available from Invitrogen as a 100 mM stock solution page viii e Opti MEM I Reduced Serum Medium pre warmed to 37 C page viii e Fetal bovine serum FBS page viii e Complete growth medium without antibiotics i e D MEM containing 10 FBS 4 mM L Glutamine 0 1 mM MEM Non Essential Amino Acids and 1 mM MEM sodium pyruvate pre warmed to 37 C e Sterile 10 cm tissue culture plates one each for the lentiviral construct positive control and negative control e Sterile tissue culture supplies e 15 mlsterile capped conical tubes e Cryovials e CO humidified incubator set at 37 C e Centrifuge capable of 2 000 x g e
75. using HT1080 cells we usually plate 2 x 10 cells per well in a 6 well plate 2 On the day of transduction Day 1 thaw your lentiviral stock and prepare 10 fold serial dilutions ranging from 10 to 10 For each dilution dilute the lentiviral stock into complete culture medium to a final volume of 1 ml DO NOT vortex Note You may prepare a wider range of serial dilutions 10 to 10 if desired 3 Remove the culture medium from the cells Mix each dilution gently by inversion and add to one well of cells total volume 1 ml 4 Add Polybrene if desired see page 17 to each well to a final concentration of 6 ug ml Swirl the plate gently to mix Incubate at 37 C overnight in a humidified 5 CO incubator 5 The following day Day 2 remove the media containing virus and replace with 2 ml of complete culture medium Incubate at 37 C overnight in a humidified 5 CO incubator 6 The following day Day 3 remove the medium and replace with complete culture medium containing the appropriate amount of Blasticidin to select for stably transduced cells Replace medium with fresh medium containing antibiotic every 4 5 days After 10 12 days of selection day 14 16 you should see no live cells in the mock well and discrete antibiotic resistant colonies in one or more of the dilution wells Remove the medium and wash the cells twice with PBS 9 Add crystal violet solution 1 ml for 6 well dish 5 ml for 10 cm plate and incubat
76. using a range of MOI e g 0 0 5 1 2 5 10 to determine the MOI required to obtain the optimal expression of your protein for your application Continued on next page Transduction and Analysis Continued N N D L1 5 V _ o E Positive Control Note Materials Needed In general we have found that 80 9076 of the cells in an actively dividing cell line e g HT1080 express a target gene when transduced at an MOI of 1 Some non dividing cell types transduce lentiviral constructs less efficiently For example only about 50 of the cells in a culture of primary human fibroblasts express a target gene when transduced at an MOI of 1 If you are transducing your lentiviral construct into a non dividing cell type you may need to increase the MOI e g MOI 10 to achieve optimal expression levels for your recombinant protein Control lentiviral vectors expressing lacZ are available for optimization see your vector manual and page viii for information If you have generated a lentiviral stock of a lacZ expression control pLenti6 3 V5 GW lacZ or pLenti6 3 V5 GW EmGFP we recommend using the stock to help you determine the optimal MOI for your particular cell line and application Once you have transduced the control lentivirus into your mammalian cell line of choice the gene encoding B galactosidase will be constitutively expressed and can be easily assayed refer to the expression vector or expression contro
77. ust be maintained in medium containing Geneticin page viii For more information about pCMVSPORT6TAg neo and how to culture and maintain 293FT cells refer to the 293FT Cell Line manual This manual is supplied with the ViraPower HiPerform Lentiviral Expression kits and is also available by downloading from www invitrogen com or by contacting Technical Support page 40 Note The 293FT Cell Line is also available separately from Invitrogen page viii The health of your 293FT cells at the time of transfection has a critical effect on the success of lentivirus production Use of unhealthy cells will negatively affect the transfection efficiency resulting in production of a low titer lentiviral stock For optimal lentivirus production i e producing lentiviral stocks with the expected titers follow the guidelines below to culture 293FT cells before use in transfection e Ensure that cells are healthy and greater than 90 viable e Subculture and maintain cells in complete medium containing 0 1 mM MEM Non Essential Amino Acids 4 mM L Glutamine 1 mM sodium pyruvate 500 pg ml Geneticin and 10 fetal bovine serum that is not heat inactivated page viii e Do not allow cells to overgrow before passaging e Use cells that have been subcultured for less than 16 passages Continued on next page Producing Lentivirus in 293FT Cells Continued Recommended Transfection Conditions Recommended Procedure We pr
78. www invitrogen com or contact Technical Support see page 40 Many of the reagents supplied in the ViraPower HiPerform Lentiviral Expression Kits as well as other products suitable for use with the kits are available separately from Invitrogen Ordering information for these reagents is provided below Item Quantity Catalog no pLenti6 3 V5 TOPO TA Cloning Kit 20 reactions K5315 20 pLenti7 3 V5 TOPO TA Cloning Kit 20 reactions K5325 20 pLenti6 3 V5 DEST Gateway Vector Kit 6 ug V533 06 pLenti7 3 V5 DEST Gateway Vector Kit 6 ug V534 06 Vivid Colors pLenti6 3 V5 GW EmGFP 20 pg V370 06 Expression Control Vector PureLink HiPure Plasmid Midiprep Kit 25 reactions K2100 04 50 reactions K2100 05 One Shot Stb13 Chemically Competent 20 x 50 ul C7373 03 E coli 293FT Cell Line 3 x 106 cells frozen R700 07 Lipofectamine 2000 0 75 ml 11668 027 1 5 ml 11668 019 Opti MEM I Reduced Serum Medium 100 ml 31985 062 500 ml 31985 070 Dulbecco s Modified Eagle Medium 500 ml 11965 092 DENE 1000 ml 11965 084 TrypLE Select 1X liquid 500 ml 12563 029 TrypLE Select Animal Origin Free 100 ml 12563 011 Trypsin Like Enzyme Propidium Iodide 100 mg P 3566 Blasticidin 50 mg R210 01 Geneticin 20 ml 10131 035 100 ml 10131 027 Fetal Bovine Serum FBS Certified 500 ml 16000 044 Phosphate Buffered Saline PBS pH 7 4 500 ml 10010 023 1L 10010 031
79. y after transfection remove media containing DNA lipid complexes and replace with media containing sodium pyruvate Sodium pyruvate provides an extra energy source for the cells Viral supernatant harvested too early Viral supernatants can generally be collected 48 72 hours posttransfection If many cells are still attached to the plate and look healthy at this point wait an additional 24 hours before harvesting the viral supernatant Harvest no later than 72 hours post transfection Viral supernatant too dilute Concentrate your virus Yee 1999 Viral supernatant frozen and thawed multiple times Do not freeze thaw viral supernatant more than 3 times Poor choice of titering cell line Use HT1080 cells or another adherent cell line with the characteristics discussed on page 17 30 Continued on next page Troubleshooting Continued Generating the Lentiviral Stock continued Problem Reason Solution Low viral titer continued Gene of interest is toxic to cells Do not generate constructs containing activated oncogenes or harmful genes Gene of interest is large Viral titers generally decrease as the size of the insert increases Concentrate the virus if titer is low see page 15 Inserts larger than 5 6 kb are not recommended Polybrene not included during transduction Transduce the lentiviral construct into cells in the presence of Polybrene
80. ys While the quantity of cells expressing your gene of interest is significantly greater than other pLenti vectors that do not contain the WPRE and cPPT elements the signal intensity of EmGFP expressed in your cells is not optimal for viewing with fluorescence microscopy For this reason we recommend flow cytometry Continued on next page Titering Your Lentivirus Stock Using EmGFP Continued Materials Needed Trypsin Dissociation Solution Transduction and Titering Procedure for EmGFP You will need the following items Your EmGFP lentiviral stock from either the pLenti7 3 V5 TOPO vector or the pLenti7 3 V5 DEST Gateway vector store at 80 C until use Adherent mammalian cell line of choice Complete culture medium for your cell line 6 mg ml Polybrene optional see page 17 96 well tissue culture plates Optional TrypLE page viii Tripsin cell dissociation solution using see below or equivalent for flow cytometry Optional Flow cytometry buffer of choice such as calcium magnesium free Phosphate Buffered Saline containing 1 FBS or BSA Before proceeding to analysis with flow cytometry you need to dissociate your cells from the wells To prepare the dissociation solution using TrypLE 1 2 Make a 1 3 mix of TrypLE and PBS respectively see page viii to order Add 25 ul of a 1 mg ml propidium iodide stock solution page viii Follow the procedure below to determine the titer of your l

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