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ViraPower™ Lentiviral Expression Systems

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1. e Includes multiple features designed to enhance the biosafety of the system continued on next page Overview continued Purpose of this Manual Components of the ViraPower Lentiviral Expression System This manual provides an overview of the ViraPower 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 BD Assay for transient expression of your recombinant protein or 5 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 Lentiviral Expression Kits and are also available for downloading from www invitrogen com or by contacting Technical Service see page 36 The ViraPower Lentiviral Expression System facilitates highly efficient in vitro or in vivo 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 Lent
2. Introduction Recommended Transfection Conditions 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 The following section provides protocols and instructions to generate a lentiviral stock We produce lentiviral stocks in 293FT cells using the following optimized transfection conditions 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 Amount Tissue culture plate size 10 cm one per lentiviral construct Number of 293FT cells to transfect 6 x 10 cells see Recommendation on page 8 to prepare cells for transfection TM Amount of ViraPower Packaging Mix 9 ug 9 ul 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 o
3. Permits high copy replication and maintenance in E coli Ampicillin bla resistance gene Allows selection of the plasmid in E coli 35 Technical Service 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 service 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 page 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_service invitrogen com E mail jpinfo invitrogen com E mail eurotech invitrogen com Material Data Safety Sheets MSDSs Limited Warranty 36 MSDSs Material Safety Data Sheets are available on our website at www invitrogen com msds
4. OH MW 1 535 Zeocin concentrations ranging from 50 1000 ug ml are typically used for efficient selection in mammalian cells Mulsant et al 1988 Zeocin is supplied in autoclaved deionized water in 1 25 ml aliquots at a concentration of 100 mg ml Handle Zeocin using the following guidelines TM e Store Zeocin at 20 C and thaw on ice before use e Zeocin is light sensitive Store the drug and medium containing drug in the dark at 4 C Culture medium containing Zeocin may be stored at 4 C protected from exposure to light for up to 1 month e Wear gloves a laboratory coat and safety glasses or goggles when handling Zeocin containing solutions e Zeocin is toxic Do not ingest or inhale solutions containing the drug 29 Map and Features of pLP1 pLP1 Map 30 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 Service see page 36 Comments for pLP1 8889 nucleotides CMV promoter bases 1 747 TATA box bases 648 651 Human globin intron bases 880 1320 HIV 1 gag pol sequences bases 1355 5661 gag coding sequence bases 1355 2857 gag pol frameshift base 2650 pol coding seq
5. gently and incubate for 5 minutes at room temperature c After the 5 minute incubation combine the diluted DNA with the diluted Lipofectamine 2000 Mix gently d Incubate for 20 minutes at room temperature to allow the DNA TM Lipofectamine 2000 complexes to form The solution may appear cloudy but this will not impede the transfection Add the DNA Lipofectamine 2000 complexes dropwise to each plate of cells Mix gently by rocking the plate back and forth Incubate the cells overnight at 37 C in a humidified 5 CO incubator The next day Day 3 remove the medium containing the DNA Lipofectamine 2000 complexes and replace with 10 ml complete culture medium without antibiotics Incubate at 37 C in a humidified 5 CO incubator 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 Harvest virus containing supernatants 48 72 hours posttransfection Day 4 5 by removing medium into to a 15 ml sterile capped conical tube Minimal differences in viral yield are observed whether supernatants are collected at either 48 or 72 hours posttransfection Caution Remember that you are working with infectious virus at this stage Follow the recommended guidelines for working with BL 2 organisms see page 4 for more information Centrifuge superna
6. 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 Zeocin Zeocin Molecular Weight Formula and Structure Zeocin Selection Handling Zeocin Zeocin belongs to a family of structurally related bleomycin phleomycin type antibiotics isolated from Streptomyces Antibiotics in this family are broad spectrum antibiotics that act as strong antibacterial and antitumor drugs They show strong toxicity against bacteria fungi including yeast plants and mammalian cells Baron et al 1992 Drocourt et al 1990 Mulsant et al 1988 Perez et al 1989 The Zeocin resistance protein has been isolated and characterized Calmels et al 1991 Drocourt et al 1990 This protein the product of the Sh ble gene Streptoalloteichus hindustanus bleomycin gene is a 13 7 kDa protein that binds Zeocin and inhibits its DNA strand cleavage activity Expression of this protein TM in eukaryotic and prokaryotic hosts confers resistance to Zeocin The formula for Zeocin is CsoHgsN210253 and the molecular weight is 1 535 The diagram below shows the structure of Zeocin H CONH2 acy Sodus ie eae cw FR NH
7. C pLenti6 2 V5 DEST Gateway Vector Kit Dry ice e Vectors 20 C e One Shot Stb13 Chemically Competent E coli 80 C pLenti6 2 C Lumio V5 DEST Vector Dry ice e Vectors 20 C e One Shot Stb13 Chemically Competent E coli 80 C pLenti6 2 N Lumio V5 DEST Vector Dry ice e Vectors 20 C e One Shot Stb13 Chemically Competent E coli 80 C pLenti6 UbC V5 DEST Gateway Vector Kit Dry ice e Vectors 20 C e One Shot Stb13 Chemically Competent E coli 80 C ViraPower Packaging Mix Room 20 C temperature continued on next page Kit Contents and Storage continued Expression Vectors ViraPower Bsd Lentiviral Support Kit Contents ViraPower Zeo Lentiviral Support Kit Contents Each ViraPower or ViraPower II Lentiviral Expression Kit also includes a pLenti based expression vector kit The expression vector kit includes e A pLenti based expression vector for cloning your gene of interest e A corresponding expression control plasmid e One Shot Stb13 Chemically Competent E coli for transformation Expression vectors include pLenti6 V5 D TOPO pLenti4 V5 DEST pLenti6 V5 DEST pLenti6 2 V5 DEST pLenti6 2 C Lumio V5 DEST pLenti6 2 N Lumio V5 DEST and pLenti6 UbC V5 DEST 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 co
8. Incubate at 37 C overnight in a humidified 5 CO incubator The following day Day 4 treat cells as follows e For Blasticidin selection remove the medium and replace with complete culture medium containing the appropriate amount of Blasticidin to select for stably transduced cells e For Zeocin selection remove the medium and wash the cells once with PBS For each well of cells trypsinize the cells and replate the entire amount into one 10 cm plate containing complete culture medium with the appropriate amount of Zeocin to select for stably transduced cells Replace medium with fresh medium containing antibiotic every 3 4 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 Add crystal violet solution 1 ml for 6 well dish 5 ml for 10 cm plate and incubate for 10 minutes at room temperature Remove the crystal violet stain and wash the cells with PBS Repeat wash Count the blue stained colonies and determine the titer of your lentiviral stock continued on next page Titering Your Lentiviral Stock 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 con
9. 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 Service 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 regarding the contents of any publications or documentation If you discover an error in any of our publications please report it to our Technical Service Representatives Invitrogen assumes no responsibility or liability for any special incidental indirect or consequential loss or damage whats
10. and target validation applications in laboratory animals 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 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 devel
11. contact Licensing Blasticidin Selection Marker Limited Use Label License No 86 Zeocin Selection Marker Limited Use Label License No 108 Lentiviral Technology 40 Department Invitrogen Corporation 1600 Faraday Avenue Carlsbad California 92008 Phone 760 603 7200 Fax 760 602 6500 The ble resistance gene is the subject of one or more of U S Patent Nos 5 021 344 and 5 118 620 and foreign equivalents owned by Cayla and licensed to Invitrogen This product is sold for research purposes only For commercial license information please contact Licensing Department Invitrogen Corporation 1600 Faraday Avenue Carlsbad CA 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
12. 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 constructs 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 sensitivit
13. of the expression plasmid and the plasmids in the packaging mix e Control expression plasmid to optimize virus production and cell transduction containing either The lacZ gene which when packaged into virions and transduced into a mammalian cell line expresses B galactosidase included with each expression vector or 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 ix for ordering information 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 TM Use of the ViraPower Lentiviral Expression System to facilitate lentiviral based expression of the gene of interest provides the following advantages 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
14. required to obtain the optimal expression of your protein for your application continued on next page Transduction and Analysis continued EN N PUE Sy Ya SE O 2 gt Positive Control Note Materials Needed In general we have found that 80 90 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 or EmGFP are available for optimization see your vector manual and page ix for information If you have generated a lentiviral stock of a lacZ expression control e g pLenti6 V5 GW lacZ 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 galactosidase or EmGFP will be constitutively expressed and can be easily assayed refer to the expression vector or expression control vector manual for assay methods Viral supernatants are genera
15. transfect 293FT cells offers the following advantages e Provides the highest transfection efficiency in 293FT cells TM 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 Lentiviral Support Kits see page ix for ordering information To facilitate optimal formation of DNA Lipofectamine 2000 complexes we recommend using Opti MEM I Reduced Serum Medium available from Invitrogen see page ix for ordering information If you producing lentivirus for the first time using the ViraPower System and 293FT cells you should perform the Forward Transfection procedure on page 12 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 called the Alternate Transfection Method If you 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 13 In this procedure 293FT cells are added to media containing the DNA Lipofectamine 2000 complexes Producing Lentivirus in 293FT Cells
16. 20 ViraPower Bsd Lentiviral Support Kit ViraPower Zeo Lentiviral Support Kit pLenti6 V5 Directional TOPO Cloning Kit pLenti4 V5 DEST Gateway Vector Kit pLenti6 V5 DEST Gateway Vector Kit pLenti6 2 V5 DEST Gateway Vector Kit pLenti6 2 N and C Lumio V5 DEST Vectors pLenti6 UbC V5 DEST Gateway Vector Kit 293FT Cell Line Y v Y Y Y Y Y continued on next page Kit Contents and Storage continued Shipping Storage vi The ViraPower Lentiviral products are shipped as described below Upon receipt store each component as detailed below Item Shipping Storage ViraPower Bsd Lentiviral Support Kit Blue ice e ViraPower Packaging Mix 20 C e Lipofectamine 2000 4 C do not freeze e Blasticidin 20 C ViraPower Zeo Lentiviral Support Kit Blue ice e ViraPower Packaging Mix 20 C e Lipofectamine 2000 4 C do not freeze e Zeocin 20 C protected from light 293FT Cell Line Dry ice Liquid nitrogen pLenti6 V5 Directional TOPO Cloning Kit Dry ice e pLenti6 V5 D TOPO Reagents 20 C e One Shot Stb13 Chemically Competent E coli 80 C pLenti4 V5 DEST Gateway Vector Kit Dry ice e Vectors 20 C e One Shot Stb13 Chemically Competent E coli 80 C pLenti6 V5 DEST Gateway Vector Kit Dry ice e Vectors 20 C e One Shot Stb13 Chemically Competent E coli 80
17. 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 otherwise 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 Com
18. 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 Too little antibiotic used for cells confluent selection Increase amount of antibiotic Zeocin selection performed on confluent cells Before adding selective medium trypsinize transduced cells and replate in a larger tissue culture plate Viral supernatant not diluted sufficiently Titer lentivirus using a wider range of 10 fold serial dilutions e g 10 to 10 Transducing The table below lists some potential problems and possible solutions that may Mammalian Cells help you troubleshoot your transduction and expression experiment Problem Reason Solution No expression of the Promoter silencing gene of interest e Lentiviral constructs may integrate into a chromosomal region that silences the CMV promoter Screen multiple antibiotic resistant clones and select the one with the highest expression levels e Use pLenti6 UbC V5 DEST to generate your lentiviral construct containing a cellular promoter that is not subject to silencing Viral stocks stored incorrectly Aliquot and store stocks at 80 C Do not freeze thaw more than 3 times 26 continued on next page Troubleshooting continued Transducing Mammalian Cells
19. E E 34 Technical AV A RE a E AE A EE 36 Purchaser Notifica z siosaia e e E S A S oaiae slices aa AES EEEL 37 References iia tt 43 iii iv Kit Contents and Storage Types of Kits TM This manual is supplied with the kits listed below The ViraPower Lentiviral TM Support Kits includes the ViraPower Packaging Mix Lipofectamine 2000 and a selection agent The ViraPower and ViraPower II Lentiviral Expression Kits TM include the ViraPower Lentiviral Support Kit plus an expression vector and the 293FT producer cell line Product Catalog no ViraPower Lentiviral Directional TOPO Expression Kit K4950 00 ViraPower Lentiviral Gateway Expression Kit K4960 00 ViraPower II Lentiviral Gateway Expression Kit K367 20 ViraPower II Lentiviral C Lumio Gateway Expression Kit K370 20 ViraPower II Lentiviral N Lumio Gateway Expression Kit K371 20 ViraPower Bsd Lentiviral Support Kit K4970 00 ViraPower Lentiviral Packaging Mix K4975 00 ViraPower Zeo Lentiviral Gateway Expression Kit K4980 00 ViraPower Zeo Lentiviral Support Kit K4985 00 ViraPower UbC Lentiviral Gateway Expression Kit K4990 00 System Components The following table shows the components associated with Lentiviral Expression Kit catalog numbers listed above Components Catalog no K4950 00 K4960 00 K4980 00 K4990 00 K367 20 K370 20 K371
20. 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 Service see page 36 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 32 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 pUC origin of replication ori Permits high copy replication and maintenance in E coli 33 Map and Features
21. Packaging Mix resuspend the contents of one tube 195 ug in 195 pl of sterile water to obtain a 1 ug nl stock TM Note ViraPower Packaging Mix is available separately from Invitrogen or as part of the ViraPower Lentiviral Support Kits page ix TM The human 293FT Cell Line is supplied with the ViraPower 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 pCMVSPORT6TAg neo and must be maintained in medium containing Geneticin For more information about pCMVSPORT6TA g neo and how to culture and maintain 293FT cells refer to the 293FT Cell Line manual This manual is supplied with the ViraPower Lentiviral Expression kits and is also available for downloading from www invitrogen com or by calling Technical Service see page 36 Note The 293FT Cell Line is also available separately from Invitrogen page ix 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 Make sure that c
22. You will use at least one 6 well plate for every lentiviral stock to be titered one mock well plus five dilutions Note If you have generated a lentiviral stock of a lacZ expression control e g pLenti6 V5 GW lacZ we recommend titering this stock as well If you are using pLenti6 2 GW EmGEFP refer to the user manual for the vector for the titering protocol 1 10 11 The day before transduction Day 1 trypsinize and count the cells plating them in a 6 well plate such that they will be 30 50 confluent at the time of transduction Incubate cells at 37 C overnight in a humidified 5 CO incubator Example When using HT1080 cells we usually plate 2 x 10 cells per well in a 6 well plate On the day of transduction Day 2 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 107 to 10 if desired Remove the culture medium from the cells Mix each dilution gently by inversion and add to one well of cells total volume 1 ml Add Polybrene if desired 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 The following day Day 3 remove the media containing virus and replace with 2 ml of complete culture medium
23. acturing 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 760 602 6500 Email outlicensing invitrogen com continued on next page 37 Purchaser Notification continued Limited Use Label License No 19 Gateway Cloning Products Gateway Clone Distribution Policy 38 This product and its use is the subject of one or more of U S Patent Nos
24. be successfully transduced with your lentivirus Follow the instructions below to prepare Polybrene Sigma Catalog no H9268 1 Prepare a 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 continued on next page Titering Your Lentiviral Stock continued Materials Needed To determine the titer of your lentiviral construct you should have the following materials before beginning Your pLenti lentiviral stock store at 80 C until use Adherent mammalian cell line of choice Complete culture medium for your cell line 6 mg ml Polybrene if desired 6 well tissue culture plates 10 cm tissue culture plates for Zeocin selection only Blasticidin 10 mg ml stock or Zeocin 100 mg ml stock as appropriate for selection Crystal violet Sigma Catalog no C3886 prepare a 1 crystal violet solution in 10 ethanol Phosphate Buffered Saline PBS Invitrogen Catalog no 10010 023 continued on next page 19 Titering Your Lentiviral Stock continued Transduction and Titering Procedure 20 Follow the procedure below to determine the titer of your lentiviral stock using the mammalian cell line of choice
25. ber 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 Cytomegalovirus 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 Calmels T Parriche M Burand H and Tiraby G 1991 High Efficiency Transformation of Tolypocladium geodes Conidiospores to Phleomycin Resistance Curr Genet 20 309 314 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 Drocourt D Calmels T P G Reynes J P Baron M and Tiraby G 1990 Cassettes of the Streptoalloteichus hindustanus ble Gene for Transformation of Lower and Higher Eukaryotes to Phleomycin Resistance Nucleic Acids Res 18 4009 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 Em
26. centrated virus transducing units TU ml In this experiment a Lenti6 V5 GW lacZ lentiviral stock was generated using the protocol on page 12 and was concentrated by ultracentrifugation HT1080 cells were transduced with 10 fold serial dilutions of the lentiviral supernatant 107 to 10 dilutions or untransduced mock following the protocol on page 20 At 48 hours post transduction the cells were placed under Blasticidin selection 10 ug ml After 10 days of selection the cells were stained with crystal violet see plate below and colonies were counted 10 102 mock 10 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 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 the Troubleshooting section page 25 for more tips and guidelines to optimize your viral yield 21 Transductio
27. 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 lowa Research Foundation and is sold for research use only Commercial users must obtain a license to these patents directly from the University of lowa Research Foundation UIRF 214 Technology Innovation Center Iowa City lowa 52242 For further information please contact the Associate Director of UIRF at 319 335 4546 continued on next page 39 Purchaser Notification continued Limited Use Label License No 51 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 Blasticidin and the purchasing a license to this product for purposes other than research
28. commend 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 performing 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 The pLenti expression constructs contain either the Blasticidin resistance gene bsd Kimura et al 1994 or the Zeocin resistance gene Calmels et al 1991 Drocourt et al 1990 to allow for Blasticidin selection Takeuchi et al 1958 Yamaguchi et al 1965 or Zeocin selection Mulsant et al 1988 respectively of mammalian cells that have stably transduced the lentiviral construct If you have purchased a ViraPower Lentiviral Expression Kit either Blasticidin or Zeocin is supplied Blasticidin and Zeocin are also available separately from Invitrogen or as part of the appropriate ViraPower Lentiviral Support Kit see page ix for ordering information Cell density can affect the efficiency of Zeocin selection For the most efficient Zeocin selection cells should not be greater than 50 confluent Fo
29. control and negative control Sterile tissue culture supplies 15 ml sterile capped conical tubes Optional Millex HV 0 45 um PVDF filters Millipore cat no SLHVR25LS or equivalent Cryovials continued on next page 11 Producing Lentivirus in 293FT Cells continued Forward Transfection Procedure 12 If you are a first time user follow the procedure below to cotransfect 293FT cells TM We recommend including a 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 On the day of transfection Day 2 remove the culture medium from the 293FT cells and replace with 5 ml of growth medium or Opti MEM I Medium containing serum Do not include antibiotics in the medium For each transfection sample prepare DNA Lipofectamine 2000 complexes as follows TM a 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 TM b Ina separate sterile 5 ml tube mix Lipofectamine 2000 gently before use then dilute 36 ul in 1 5 ml of Opti MEM I Medium without serum Mix
30. d look healthy at this point wait an additional 24 hours before harvesting the viral supernatant 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 continued on next page 25 Troubleshooting continued Generating the Lentiviral Stock continued Problem Reason Solution Low viral titer Gene of interest is toxic to cells Do not generate constructs containing continued 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 14 Inserts larger than 5 6 kb are not recommended Polybrene not included during Transduce the lentiviral construct into cells in transduction the presence of Polybrene Lipofectamine 2000 handled e Store at 4 C Do not freeze incorrectly e Mix gently by inversion Do not vortex No colonies obtained Too much antibiotic used for upon titering 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
31. de 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 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
32. ells 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 ug ml Geneticin and 10 fetal bovine serum that is not heat inactivated page ix e Do not allow cells to overgrow before passaging e Use cells that have been subcultured for less than 16 passages General Information continued Positive Control Lipofectamine 2000 Opti MEM Recommended Procedure 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 Each pLenti expression vector kit includes a positive control vector for use as an expression control e g pLenti6 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 pLenti6 2 GW EmGFP containing Emerald Green Fluorescent Protein EmGFP for fluorescent detection is available separately from Invitrogen For ordering information see page ix TM 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
33. ent Infection of Primary Hepatocytes Proc Natl Acad Sci USA 91 9564 9568 Yee J K 1999 in The Development 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 2002 2006 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 44 8 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
34. fety The ViraPower Lentiviral Expression System includes the following key safety Features of the features ViraPower e The pLenti expression vector contains a deletion in the 3 LTR AU3 that does Lentiviral System 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 progeny 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 actua
35. i 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 Mammalian 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 Mulsant P Tiraby G Kallerhoff J and Perret J 1988 Phleomycin Resistance as a Dominant Selectable Marker in CHO Cells Somat Cell Mol Genet 14 243 252 Naldini L 1998 Lentiviruses as Gene Transfer Agen
36. iew continued How Lentivirus Works VSV Envelope Glycoprotein 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 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 Most retroviral vectors are limited in their usefulness as gene delivery vehicles by their restricted tropism and generally low titers In the ViraPower 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 Biosafety Features of the System Introduction The ViraPower Lentiviral Expression System is a third generation system based on lentiviral vectors developed by Dull et al 1998 This third generation 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 Biosa
37. ill not impede the transfection 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 Add the DNA Lipofectamine 2000 complexes 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 Add 5 ml of the 293FT cell suspension 6 x 10 total cells to the plate containing media and DNA Lipofectamine 2000 complexes Mix gently by rocking the plate back and forth Incubate cells overnight at 37 C in a humidified 5 CO incubator The next day Day 2 remove the medium containing the DNA Lipofectamine 2000 complexes and replace with 10 ml complete culture medium without antibiotics Incubate cells overnight at 37 C in a humidified 5 CO incubator 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 Harvest virus containing supernatants 48 72 hours posttransfection Day 3 4 by removing medium into a 15 ml sterile capped conical tube Minimal differences in viral yield are observed whether supernatants are collected 48 or 72 hours posttransfection Caution Remember that you are work
38. ing with infectious virus at this stage Follow the recommended guidelines for working with BL 2 organisms see page 4 for more information Centrifuge supernatants at 3000 rpm 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 Pipet viral supernatants into cryovials in 1 ml aliquots Store viral stocks at 80 C Proceed to Titering Your Lentiviral Stock page 15 continued on next page 13 Producing Lentivirus in 293FT Cells continued If you plan to use your lentiviral construct for in vivo applications we recommend Note 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 Concentrating It is possible to concentrate VSV G pseudotyped lentiviruses using a variety of Virus methods without significantly affecting their ability to transduce cells If your cell transduction experiment requires that you use a relatively high MOI you may wish to concentrate your virus before titering and proceeding to transduction For details and guidelines to concentrate your virus supe
39. invitrogen ViraPower Lentiviral Expression Systems Lentiviral systems for high level expression in dividing and non dividing mammalian cells Catalog nos K4950 00 K4960 00 K4970 00 K4975 00 K4980 00 K4985 00 K4990 00 K367 20 K370 20 and K371 20 Version G 14 April 2006 25 0501 ii Table of Contents Kit Contents nd traia oia v Accessory OO A A NOS ix Product Qualification A a Se ee oes x Introduction anne 1 AA A OA ON 1 Biosafety Features of the System oooconicononcoooonnonononenonnnncnnarannnnnnnonnnnananannnnnnnnnnnnannnnnnnnn nana E aea ae AREER EA EERE 4 Experimental Outline viii iio ted ah as oe sate aah besos Re IRB ein ath be ae ents eet 6 Methods ee 7 General Information totali el nia aaa 7 Producing Lentivirus in 293FT Cells ccccccssssssesesssesesceseeeneesesesesesnssnesesssesnansnssescscsesceceeeuenenesesescsceceeseeeneneiees 10 Titering Your Lentiviral Stock senie ei e eas Ei iat E RE E E e EA E REE 15 Transduction and Analysis or is A soi E AAA E AA ii 22 en on E unin an serie waar ian Ann eam 25 Troubleshooting anida oi idad Rob INRIRDD Ahr 25 BlaSticidi diia leticia iria lic adds a E Hobbies 28 ZROCN sn ES EEN ES arama aoe ESEE E narnia aa anania aed 29 Map and Features of PLET ioniene iieaeoe etier neetako nieno NE nEn ER eSEE ee E neenon PEEKE oiea sinai 30 Map and Features o p GPZ iyii ii ai Adi oil 32 Map and Featurres 0f PLP VSVG omisit en ior SEN RE Ae EEEE doni EREE EE EEE oat E EAEE T
40. is 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 manufacture of such product If the purchaser is not willing to accept the limitations of this limited use statement Invitrogen i
41. iviral 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 A pLenti based expression vector into which the gene of interest will be cloned 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 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 30 34 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 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 will be used to transduce a mammalian cell line of interest continued on next page Overv
42. llowing transduction and selection If you wish to select for stably transduced cells you must first determine the minimum concentration of Blasticidin or Zeocin as appropriate required to kill your untransduced mammalian cell line i e perform a kill curve experiment For guidelines to perform a kill curve experiment see page 17 If you titered your lentiviral construct in the same mammalian cell line that you are using to perform your stable expression experiment then you may use the same concentration of Blasticidin or Zeocin for selection that you used for titering 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 MOT 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 using a range of MOI e g 0 0 5 1 2 5 10 to determine the MOI
43. lly 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 exercise extra caution when creating lentivirus carrying potential harmful or toxic genes e g activated oncogenes For more information about the BL 2 guidelines and lenti
44. m that of other common antibiotics such as Blasticidin or Geneticin Zeocin sensitive cells do not round up and detach from the plate but may exhibit the following morphological changes e Vast increase in size similar to the effects of cytomegalovirus infecting permissive cells e Abnormal cell shape e Presence of large empty vesicles in the cytoplasm breakdown of the endoplasmic reticulum and Golgi apparatus or scaffolding proteins e Breakdown of plasma and nuclear membrane appearance of many holes in these membranes Eventually these cells will completely break down and only strings of protein will remain Zeocin resistant cells should continue to divide at regular intervals to form distinct colonies There should not be any distinct morphological changes in Zeocin resistant cells when compared to non selected cells Lentivirus transduction may be enhanced if cells are transduced in the presence of hexadimethrine bromide Polybrene 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 ug 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
45. mercial 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 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 an
46. n and Analysis Introduction Important Transient vs Stable Expression Determining Antibiotic Sensitivity for Your Cell Line Multiplicity of Infection MOI Determining the Optimal MOI 22 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 Pool a heterogeneous population of cells and test for expression directly after transduction i e transient expression Note that you must wait fora minimum of 48 72 hours after transduction before harvesting your cells to allow expressed protein to accumulate in transduced cells e Select for stably transduced cells using Blasticidin or Zeocin as appropriate This requires a minimum of 10 12 days after transduction but allows generation of clonal cell lines that stably express the gene of interest Note We have observed stable expression of a target gene for at least 6 weeks fo
47. ned 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 gt 4 kb you will need to concentrate the virus to obtain a suitable titer see page 14 The size of the wild type HIV genome 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 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 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 Number of freeze thaw cycles Viral titers can decrease as much as 10 with each freeze thaw cycle 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 Antibiotic Selection Important Preparing Blasticidin or Zeocin Determining Antibiotic Sensitivity We strongly re
48. ntaining attB1 and attB2 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 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 References Andersson S Davis D L Dahlb ck 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 We
49. ntaining your gene of interest TM The ViraPower Bsd 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 Amount Storage ViraPower Packaging Contains a mixture of the pLP1 195 ug 20 C Mix pLP2 and pLP VSVG plasmids lyophilized in TE pH 8 0 Lipofectamine 2000 Proprietary 0 75 ml 4 C Blasticidin Powder 50 mg 20 C TM The ViraPower Zeo Lentiviral Support Kit includes the following vectors and reagents Store as directed below Important Tips e Take care to store Lipofectamine 2000 at 4 C DO NOT FREEZE e Zeocin is light sensitive Store at 20 C in the dark Reagent Composition Amount Storage ViraPower Packaging Contains a mixture of the pLP1 195 ug 20 C Mix pLP2 and pLP VSVG plasmids lyophilized in TE pH 8 0 Lipofectamine 2000 Proprietary 0 75 ml 4 C Zeocin 100 mg ml in sterile deionized 12 5 mg 20 C water protected from light continued on next page Vii Kit Contents and Storage continued ViraPower Catalog no K4975 00 contains 3 tubes with 195 ug DNA per tube of ViraPower Packaging Mix Packaging Mix Upon receipt store at 20 C 293FT Cell Line Each ViraPower Lentiviral Expression Kit includes the 293FT producer cell line The 293FT Cell Line i
50. ocin prevents growth of the E coli strain TOP10 Each lot of cells is tested for cell growth and viability post recovery from cryopreservation Master Cell Banks are screened for viruses mycoplasma and sterility and expression of the SV40 large T antigen is confirmed by western blot TM Using the reagents provided in the kit the ViraPower Packaging Mix and a control lentiviral construct are cotransfected into 293FT cells using the protocol on page 12 Lentiviral supernatants are harvested 48 hours posttransfection and the titer is determined using HT1080 cells The control lentiviral construct must demonstrate a titer of greater than 1 x 10 TU ml Overview Introduction Advantages of the System Introduction TM The ViraPower Lentiviral Expression System allows 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 major components of the system include e An expression plasmid containing the gene of interest under the control of a choice of promoters and elements that allow packaging of the construct into virions e 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 e The 293FT cell line which allows production of lentivirus following cotransfection
51. oever 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 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 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 manuf
52. of pLP VSVG pLP VSVG Map 34 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 Service see page 36 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 globin polyadenylation signal Allows efficient transcription termination and polyadenylation of mRNA pUC origin of replication ori
53. oped 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 continued on next page Purchaser Notification continued Limited Use Label License No 109 Retroviral Helper Lines Information for European Customers 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 th
54. or 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 S N A P MidiPrep Kit which contains 10 mM EDTA in the Resuspension Buffer see page ix for ordering information General Information continued ViraPower Packaging Mix 293FT Cell Line NANE Vy Y SN o P 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
55. pofectamine 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 Blasticidin 50 mg R210 01 Zeocin lg R250 01 5g R250 05 Geneticin 20 ml 10131 035 100 ml 10131 027 Phosphate Buffered Saline PBS pH 7 4 500 ml 10010 023 1L 10010 031 ix Product Qualification Introduction ViraPower Packaging Mix Lipofectamine 2000 Blasticidin Zeocin 293FT Cell Line Lentivirus Production This section describes the criteria used to qualify the components of the ViraPower Lentiviral Expression System TM Each vector in the ViraPower Packaging Mix is qualified individually prior to mixing by restriction enzyme digestion For information about how the pLenti expression vectors are qualified refer to the specific manual for each expression vector TM Lipofectamine 2000 is tested for the absence of microbial contamination using blood agar plates Sabaraud dextrose agar plates and fluid thioglycolate medium and functionally by transfection of CHO K1 cells with a reporter plasmid Blasticidin is lot qualified by performing a kill curve on Blasticidin sensitive and Blasticidin resistant mammalian cell lines Blasticidin sensitive cells should be killed at all concentrations tested 2 5 10 ug ml within 10 days after addition of Blasticidin Zeocin is lot qualified by demonstrating that LB media containing 25 ug ml Ze
56. protocols are provided in this section to titer your lentiviral stock Note If you are using pLenti6 2 GW EmGFP Expression Control Vector to produce a lentiviral stock refer to the user manual for titer methods using fluorescent detection Remember that you will be 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 lentiviral stocks you will 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 Stain and count the number of Blasticidin resistant colonies in each dilution continued on next page 15 Titering Your Lentiviral Stock continued Factors Affecting Viral Titer 16 A number of factors can influence viral titers including The size of your gene of interest Titers will decrease as the size of the insert increases We have determi
57. ptimization will be necessary to obtain the expected titers continued on next page Producing Lentivirus in 293FT Cells continued Materials Needed You should have the following materials before beginning TM ViraPower Packaging Mix supplied with the kit resuspend in 195 ul of sterile water to a concentration of 1 ug pl pLenti expression vector containing your gene of interest 0 1 3 0 ug ul in sterile water or TE pH 8 0 pLenti control vector containing lacZ supplied with the kit or EmGFP available separately resuspended in sterile water to a concentration of 1 ug ul 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 ug 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 Catalog no 11360 070 Lipofectamine 2000 transfection reagent supplied with the kit store at 4 C and mix gently before use Opti MEM I Reduced Serum Medium pre warmed to 37 C Fetal bovine serum FBS Cat no 16000 044 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 Sterile 10 cm tissue culture plates one each for the lentiviral construct positive
58. r more information about how to prepare and handle Blasticidin and Zeocin refer to the Appendix pages 25 and 29 respectively Since you will be selecting for stably transduced cells using Blasticidin or Zeocin you must first determine the minimum concentration of Blasticidin or Zeocin required to kill your untransduced mammalian cell line i e perform a kill curve experiment Typically concentrations ranging from 2 10 ug ml Blasticidin or 50 1000 ug ml Zeocin 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 2 The next day substitute culture medium with medium containing varying concentrations of Blasticidin or Zeocin as appropriate 3 Replenish the selective media every 3 4 days and observe the percentage of surviving cells 4 Determine the appropriate concentration of Blasticidin or Zeocin that kills the cells within 10 14 days after addition of antibiotic continued on next page 17 Titering Your Lentiviral Stock continued Effect of Zeocin on Sensitive and Resistant Cells Using Polybrene During Transduction Preparing and Storing Polybrene 18 Zeocin s method of killing is quite different fro
59. rnatant by ultracentrifugation refer to published reference sources Yee 1999 Long Term Store viral stocks at 80 C in cryovials for long term storage Repeated freezing Storage 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 line of interest Scaling Up Virus It is possible to scale up the cotransfection experiment to produce a larger volume Production 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 14 Titering Your Lentiviral Stock Introduction Experimental Outline 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
60. s 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 S N A P Midiprep DNA Isolation Kit 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 ug Lipofectamine 2000 in ul 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 13 Transfected cells not cultured in media containing sodium pyruvate One day 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 an
61. s if desired 5 Assay for recombinant protein of interest Methods General Information Introduction Generating Your pLenti Expression Construct DNA Isolation Guidelines Important TM The ViraPower 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 To generate a pLenti expression construct containing your gene of interest refer to the manual for the vector you are using for instructions Once you have created your expression construct you will 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 f
62. s 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 Lentiviral Expression Kit and available at www invitrogen com viii Accessory Products Introduction Additional Products The products listed in this section may be used with the ViraPower Lentiviral Expression Kits For more information go to www invitrogen com or call Technical Service see page 36 TM Many of the reagents supplied in the ViraPower 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 V5 Directional TOPO Cloning Kit 20 reactions K4955 10 pLenti4 V5 DEST Gateway Vector 6 ug V498 10 pLenti6 V5 DEST Gateway Vector 6 ug V496 10 pLenti6 2 V5 DEST Gateway Vector 6 ug V368 20 pLenti6 UbC V5 DEST Gateway Vector 6 ug V499 10 pLenti6 2 GW EmGFP Expression Control 20 ug V369 20 Vector One Shot Stb13 Chemically Competent 20 x 50 ul C7373 03 E coli S N A P Midiprep DNA Isolation Kit 20 reactions K1910 01 293FT Cell Line 3 x 10 cells frozen R700 07 Fetal Bovine Serum FBS Certified 500 ml 16000 044 Li
63. s 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 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 41 Gateway Clone Distribution Policy Introduction Gateway Entry Clones Gateway Expression Clones Additional Terms and Conditions 42 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 co
64. stock and if necessary dilute the appropriate amount of virus into fresh complete medium to obtain a suitable MOL 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 ug 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 or Zeocin as appropriate 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 da
65. tants at 3000 rpm 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 Pipet viral supernatants into cryovials in 1 ml aliquots Store viral stocks at 80 C Proceed to Titering Your Lentiviral Stock page 15 continued on next page Producing Lentivirus in 293FT Cells continued Reverse Transfection Procedure If you are an experienced user you may use the rapid procedure below to cotransfect 293FT cells 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 TM On Day 1 prepare DNA Lipofectamine 2000 complexes for each transfection sample as follows TM a 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 TM b Ina separate sterile 5 ml tube mix Lipofectamine 2000 gently before use then dilute 36 ul in 1 5 ml of Opti MEM I Medium without serum Mix gently and incubate for 5 minutes at room temperature c After the 5 minute incubation combine the diluted DNA with the diluted Lipofectamine 2000 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 w
66. ted 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 materials before beginning e Your titered lentiviral stock 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 e Appropriately sized tissue culture plates for your application e Blasticidin or Zeocin as appropriate if selecting for stably transduced cells continued on next page 23 Transduction and Analysis continued Transduction Procedure Note Detecting Recombinant Protein 24 Follow the procedure below to transduce the mammalian cell line of choice with your lentiviral construct Reminder If you are performing Zeocin selection remember that cells should not be confluent at the time of selection see Step 6 below Plate your cells accordingly 1 Plate cells in complete media as appropriate for your application 2 On the day of transduction Day 1 thaw your lentiviral
67. ts 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 continued on next page 43 References Pandya S Klimatcheva E and Planelles V 2001 Lentivirus and foamy virus vectors novel gene therapy tools Expert Opinion on Biological Therapy 1 17 40 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 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 Effici
68. uence 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 b a 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 maintenance in E coli Ampicillin bla resistance gene Allows selection of the plasmid in E coli 31
69. virus 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 Lentiviral Expression System Experimental Outline Flow Chart The diagram below describes the general steps required to express your gene of interest using the ViraPower Lentiviral Expression System Refer to the appropriate manual for each pLenti expression vector for instructions to generate your pLenti expression construct Expression Construct ViraPower Packaging Mix 293FT Producer Cell Line Your Mammalian Cell Line of Interest promoter gene of interest V5 1 Generate the pLenti expression construct containing your gene of interest 2 Cotransfect the 293FT producer cell line with your pLenti expression construct and the optimized packaging mix 3 Harvest viral supernatant and determine the titer 4 Add the viral supernatant to our mammalian cell line of interest Select for stably transduced cell
70. y 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 42 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 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 ma
71. y 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 27 Blasticidin Description Molecular Weight Formula and Structure Handling Blasticidin Preparing and Storing Stock Solutions 28 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 Formula Cy7H26NgOs5 HCl NSN A N HOOC O AAA a 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
72. ys after selection 8 Pick at least 5 antibiotic resistant colonies see Note below and expand each clone 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 Troubleshooting Appendix Generating the Lentiviral Stock The table below lists some potential problems and possible solutions that may help 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 antibiotic

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