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1. 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 37 Map and Features of pLP1 pLP1 Map 38 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 self cleaved by the viral protease into individual Gag and Pol polyproteins The sequence of pLP1 is available at www invitrogen com or by contacting Technical Support see page 45 Comments for pLP1 8889 nucleotides CMV promoter bases 1 747 TATA box bases 648 651 Human f 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 sequence bases 2650 5661 HIV 1 Rev response element RRE bases 5686 5919 Human f globin polyadenylation signal bases 6072 6837 pUC origin bases 6995 7668 C Ampicillin 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 functionally tested pLP1 8 889 bp contains the following elements All features have been Featu
2. Gene of interest is toxic to cells Try a different cell line 36 Blasticidin Description Molecular Weight Formula and Structure Handling Blasticidin Preparing and Storing Stock Solutions Appendix Blasticidin S HCl is a nucleoside antibiotic isolated from Streptomyces griseochromogenes which inhibits protein synthesis in both prokaryotic and eukaryotic cells Takeuchi et al 1958 Yamaguchi et al 1965 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 1 350 NH2 MW 458 9 Formula Ci7H26NgOs HCl SS N HOOC O A HCl a da NH NH2 O Always wear gloves mask goggles and protective clothing e g a laboratory coat when handling Blasticidin Weigh out Blasticidin and prepare solutions in a hood Blasticidin may be obtained in 50 mg aliquots see page 44 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
3. Naldini L 1998 Lentiviruses as Gene Transfer Agents for Delivery to Non dividing Cells Curr Opin Biotechnol 9 457 463 Continued on next page 54 References Continued Naldini L 1999 in The Development of Human Gene Therapy Friedmann T ed pp 47 60 Cold Spring Harbor Laboratory Press Cold Spring Harbor NY Naldini L Blomer U Gage F H Trono D and Verma I M 1996 Efficient Transfer Integration and Sustained Long Term Expression of the Transgene in Adult Rat Brains Injected with a Lentiviral Vector Proc Natl Acad Sci USA 93 11382 11388 Nelson J A Reynolds Kohler C and Smith B A 1987 Negative and Positive Regulation by a Short Segment in the 5 Flanking Region of the Human Cytomegalovirus Major Immediate Early Gene Molec Cell Biol 7 4125 4129 Pandya 5 Klimatcheva E and Planelles V 2001 Lentivirus and foamy virus vectors novel gene therapy tools Expert Opinion on Biological Therapy 1 17 40 Park F and Kay MA 2001 Modified HIV 1 based lentiviral vectors have an effect on viral transduction efficiency and gene expression in vitro and in vivo Mol Ther 4 3 164 173 Sastry L Johnson T Hobson M J Smucker B and Cornetta K 2002 Titering Lentiviral vectors comparison of DNA RNA and marker expression methods Gene Ther 9 1155 1162 Takeuchi S Hirayama K Ueda K Sakai H and Yonehara H 1958 Blasticidin S A New Antibiotic The Jo
4. 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 53 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 Weber F Jahn G Dorsch H sler K Fleckenstein B and Schaffner W 1985 A Very Strong Enhancer is Located Upstream of an Immediate Early Gene of Human 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 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
5. vector and pLenti7 3 V5 DEST Gateway vectors which are pLenti Gateway Destination vectors adapted for use with the Gateway technology e pLenti6 3 V5 TOPO vector and pLenti7 3 V5 TOPO vectors which are pLenti TOPO vectors that combine the ViraPower HiPerform Lentiviral Expression Systems with the rapid TOPO Cloning technology TM TM The new ViraPower HiPerform Lentiviral Expression vectors contain two new elements WPRE and cPPT to yield cell specific high performance results The Woodchuck Posttranscriptional Regulatory Element WPRE from the woodchuck hepatitis virus placed directly downstream of the gene of interest allows for increased transgene expression Zufferey et al 1998 with more cells expressing the gene of interest PPT Polypurine Tract from the HIV 1 integrase gene increases the copy number of lentivirus integrating into the host genome Park 2001 and allows for a two fold increase in viral titer Together WPRE and cPPT produce at least a four fold protein expression increase in most cell types compared to other vectors that do not contain these elements The ViraPower HiPerform Lentiviral FastTiter Expression Systems Cat nos K5320 00 and K5340 00 allow for an accurate titer of functional lentivirus in just two days using EMGFP TM IM TM The ViraPower HiPerform Lentiviral Expression System vectors also contain all of the following e Human cytomegalovirus CM
6. Non Essential Amino Acids 2 mM L Glutamine 1 mM sodium pyruvate 500 pg mL Geneticin and 10 fetal bovine serum that is not heat inactivated page 44 Note D MEM already contains 4 mM L Glutamine which is enough to support growth of 293FT cells However since L Glutamine slowly decays over time supplement the medium with 2 mM L Glutamine 293FT cells grow well in 6 mM L Glutamine but higher concentrations of L Glutamine may reduce growth e Use cells that have been subcultured for less than 16 passages Continued on next page Producing Lentivirus in 293FT Cells Continued Recommended Transfection Conditions Recommended Procedure We produce lentiviral stocks in 293FT cells using the optimized transfection conditions shown in the table below The amount of lentivirus produced using these recommended conditions 10 mL of virus at a titer of at least 1 x 10 transducing units TU mL is generally sufficient to transduce at least 1 x 10 cells at a multiplicity of infection MOI value of 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 ofa 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 previous page to prepare cells for transfection Amount of ViraPower Packaging M
7. bases 1916 2014 Ampicillin b a resistance gene bases 2015 2875 pUC origin bases 3020 3693 Continued on next page 40 Map and Features of pLP2 Continued Features of pLP2 4 180 bp contains the following elements All features have been pLP2 functionally 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 and on the pLenti6 BLOCK iT DEST expression vector to induce gag and pol expression which promotes 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 41 Map and Features of pLP VSVG pLP VSVG Map 42 The figure below shows the features of the pLP VSVG vector The sequence of pLP VSVG is available at www invitrogen com or by contacting Technical Support see page 45 Comments for pLP VSVG 5821 nucleotides CMV promoter bases 1 747 TATA box bases 648 651 Human B globin intron bases 880 1320 VSV G glycoprotein VSV G bases 1346 2881 Human f globin polyadenylation signal bases 3004 3769 pUC origin bases 392
8. 1 FBS at the required density for analysis on your flow cytometer Fixing the cells is not necessary but may be done see Note above 2 Use the mock transduced cells and the lowest dilution of the virus i e 107 as negative and positive samples to set up the parameters of your flow cytometer Continued on next page 23 Titering Your Lentiviral Stock Using EmGFP Continued Calculating Lentiviral Titer for EmGFP What You Can Expect 24 EmGFP lentivirus titers should be calculated from the dilutions at which the percentage of GFP positive cells fall within the range of 1 30 White et al 1999 Sastry et al 2002 This is to avoid analyzing dilution samples containing multiple integrated lentiviral genomes which may result in an underestimate of the viral titer or dilution samples containing too few transduced cells which will give inaccurate results Titer is expressed as transducing units TU mL The following formula White et al 1999 Sastry et al 2002 is used to calculate the titer F x C V xD F frequency of GFP positive cells percentage obtained divided by 100 C total number of cells in the well at the time of transduction V volume of inoculum in mL D lentivirus dilution In the following example an EmGFP lentiviral stock was generated using the protocol on the previous page The stock was concentrated and the following data were generated after performing flow cytometry Lenti
9. Focus 21 54 55 Dull T Zufferey R Kelly M Mandel R J Nguyen M Trono D and Naldini L 1998 A Third Generation Lentivirus Vector with a Conditional Packaging System J Virol 72 8463 8471 Emi N Friedmann T and Yee J K 1991 Pseudotype Formation of Murine Leukemia Virus with the G Protein of Vesicular Stomatitis Virus J Virol 65 1202 1207 Gorman C M Merlino G T Willingham M C Pastan I and Howard B H 1982 The Rous Sarcoma Virus Long Terminal Repeat is a Strong Promoter When Introduced into a Variety of Eukaryotic Cells by DNA mediated Transfection Proc Natl Acad Sci USA 79 6777 6781 Izumi M Miyazawa H Kamakura T Yamaguchi I Endo T and Hanaoka F 1991 Blasticidin S Resistance Gene bsr A Novel Selectable Marker for 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
10. Procedure no DNA no Lipofectamine 2000 in your experiment to help evaluate results First Time Users 1 The day before transfection Day 1 plate 293FT cells in a 10 cm tissue culture plate so that they will be 90 95 confluent on the day of transfection i e 5 x 10 cells in 10 mL of growth medium containing serum Do not include antibiotics in the medium Incubate the cells overnight at 37 C in a humidified 5 CO incubator 2 On the day of transfection Day 2 remove and discard the culture medium from the 293FT cells and replace it with 5 mL of growth medium Opti MEM I Medium page 44 containing serum Do not use antibiotics in the medium 3 For each transfection sample prepare DNA Lipofectamine 2000 complexes a Ina sterile 5 mL tube dilute 9 ug of the ViraPower Packaging Mix and 3 ug of your pLenti expression plasmid DNA 12 ug total in 1 5 mL of Opti MEM I Medium without serum Mix gently b Ina separate sterile 5 mL tube dilute 36 uL of Lipofectamine 2000 mix gently before use in 1 5 mL of Opti MEM I Medium without serum Mix gently and incubate for 5 minutes at room temperature c After incubation combine the diluted DNA from Step a with the diluted Lipofectamine 2000 from Step b Mix gently d Incubate the tube for 20 minutes at room temperature to allow the DNA Lipofectamine 2000 complexes to form The solution may appear cloudy but this will not impede the transfection 4 Add a
11. cells under passage 16 do not overgrow them e Although Geneticin is required for stable maintenance of 293FT cells do not add Geneticin to medium during transfection as this reduces transfection efficiency and causes cell death e Usea DNA in pg Lipofectamine 2000 in pL ratio ranging from 1 2 to 1 3 e Use more DNA Lipofectamine 2000 keeping the ratios the same For example use 5 ug of lentiviral vector 15 ug of packaging mix and 60 pL of 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 16 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 and look healthy at this point wait an additional 24 hours before harvesting the viral supernatant Harvest no later than 72 hours post transfection Viral supernatant too dilute Concentrate your virus Yee 1999 Continued on next page 33 Troubleshooting Continued Generating the Lentiviral St
12. coli strains such as Stb13 that are wild type for endonuclease 1 endA1 ensure that Solution I of the Lysis or Resuspension Buffer contains 10 mM EDTA EDTA inactivates the endonucleases and prevents DNA nicking and vector degradation Alternatively follow the instructions included the plasmid purification kits for endA1 E coli strains Do not use mini prep plasmid DNA for lentivirus production We recommend preparing lentiviral plasmid DNA using the PureLink HiPure Plasmid MidiPrep Kit which contains 10 mM EDTA in the Resuspension Buffer page 44 11 Producing Lentivirus in 293FT Cells Introduction ViraPower Packaging Mix 293FT Cell Line SANE Y Y o a 12 Before creating a stably transduced cell line expressing your gene of interest you first need to produce a lentiviral stock containing the packaged pLenti expression construct by cotransfecting the optimized packaging plasmid mix and your pLenti expression construct into the 293FT Cell Line This section provides protocols and instructions for generating a lentiviral stock The pLP1 pLP2 pLP VSVG plasmids are provided as 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 pg and Lipofectamine 2000 transfection reagent 0 75 mL supplied in the ViraPower Lentiviral Expression kit is sufficient to perform 20 cot
13. 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 Buchschacher amp Wong Staal 2000 Luciw 1996 e Retroviral and lentiviral vectors see Naldini 1999 Naldini 1998 Yee 1999 and Pandya et al 2001 We recommend including a positive control vector in your cotransfection experiment to generate a control lentiviral stock to help 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 3 V5 TOPO lacZ or pLenti6 3 V5 GW lacZ For more information about the positive control vector supplied with each kit refer to the appropriate expression vector manual e A control lentiviral expression vector containing Emerald Green Fluorescent Protein EmGFP for fluorescent detection pLenti6 3 V5 GW EmGFP is available separately page 44 Continued on next page General Information Continued Lipofectamine 2000 Opti MEM I 10 The Lipofectamine 2000 reagent supplied with the kit is a proprietary cationic lipid based formulation suitable for the transfection of nucleic acids into eukaryotic cells Ciccarone et al 1999 Using Lipofectamine 2000 to transfect 293FT cells offers the following advantages e Pro
14. or an equivalent cell dissociation solution Optional Flow cytometry buffer of choice such as calcium magnesium free Phosphate Buffered Saline containing 1 FBS or BSA Before proceeding to analysis with flow cytometry you need to dissociate your cells from the wells To prepare the dissociation solution using TrypLE de 2 TM Dilute TrypLE 1 3 in PBS Add 25 pL of a 1 mg mL propidium iodide stock solution see page 44 for ordering information Follow the procedure below to determine the titer of your lentiviral stock You will use one 96 well plate for each lentiviral stock to be titered ae 24 hours before transduction seed cells in a 96 well plate at a density of 6 000 cells per well Incubate the cells in a 37 C CO incubator overnight On the day of transduction Day 1 thaw your lentiviral stock In a biosafety cabinet prepare a 50 fold or 20 fold serial dilution of the Lentiviral stock in DMEM growth medium supplemented with Polybrene page 19 Mix each virus dilution gently by inversion DO NOT vortex Important Do NOT dilute virus in culture medium containing Blasticidin Remove the culture medium from each well of cells and replace it with the diluted virus solution Allocate 3 6 replicate wells per sample Swirl the plate gently to mix Incubate the plate at 37 C in a CO incubator overnight After 24 hours incubation Day 2 remove the virus containing medium from each well and dis
15. supplement the medium with 2 mM L Glutamine 293FT cells grow well in 6 mM L Glutamine but higher concentrations of L Glutamine may reduce growth e Opti MEM I Reduced Serum Medium pre warmed to 37 C page 44 e Fetal bovine serum FBS page 44 e Complete growth medium without antibiotics i e D MEM containing 10 FBS 2 mM L Glutamine 0 1 mM MEM Non Essential Amino Acids and 1 mM MEM sodium pyruvate pre warmed to 37 C Note MEM Sodium Pyruvate provides an extra energy source for the cells and is available separately see page 44 See note above for L Glutamine concentration e Sterile 10 cm tissue culture plates one each for the lentiviral construct positive control and negative control e Sterile tissue culture supplies e 15 mL sterile capped conical tubes e Cryovials e CO humidified incubator set at 37 C e Centrifuge capable of 2 000 x g e Optional Millex HV 0 45 um PVDF filters or equivalent e Optional pLenti control vector containing EmGFP sold separately page 44 Components supplied with the kits e ViraPower Packaging Mix e pLenti control vector containing lacZ e Lipofectamine 2000 transfection reagent mix gently before use Continued on next page 14 Producing Lentivirus in 293FT Cells Continued Forward If you are a first time user follow the procedure below to cotransfect 293FT cells Transfection For information on positive controls see page 9 Include a negative control
16. the plate at 37 C overnight in a humidified 5 CO incubator The following day Day 3 remove the medium and replace it with complete culture medium containing the appropriate amount of Blasticidin to select for stably transduced cells Replace the spent medium with fresh medium containing Blasticidin every 45 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 into each well 1 mL for 6 well plate 5 mL for 10 cm plate and incubate the plate for 10 minutes at room temperature Remove the crystal violet solution and wash the cells with PBS Repeat the wash Count the blue stained colonies and determine the titer of your lentiviral stock Continued on next page 27 Titering Your Lentiviral Stock Using Blasticidin Continued What You Should See Example of Expected Results Next Steps 28 When titering pLenti lentiviral stocks using HT1080 cells you should expect to obtain titers ranging from 1 x 10 to 5 x 10 for unconcentrated virus and up to 2 x 10 for concentrated virus transducing units TU mL In this experiment a Lenti6 3 V5 GW lacZ lentiviral stock was generated using the protocol described on page 15 and then concentrated by ultracentrifugation HT1080 cells were transduced with 10
17. www invitrogen com or contact Technical Support see page 45 Product Amount Cat no pLenti6 3 V5 TOPO TA Cloning Kit 20 reactions K5315 20 pLenti7 3 V5 TOPO TA Cloning Kit 20 reactions K5325 20 pLenti6 3 V5 DEST Gateway Vector Kit 6 ug V533 06 pLenti7 3 V5 DEST Gateway Vector Kit 6 ug V534 06 Vivid Colors pLenti6 3 V5 GW EmGFP Expression 20 ug V370 06 Control Vector ViraPower Lentiviral Packaging Mix 60 reactions K4975 00 PureLink HiPure Plasmid Midiprep Kit 25 reactions K2100 04 50 reactions K2100 05 One Shot Stb13 Chemically Competent E coli 20 x 50 pL C7373 03 293FT Cell Line 3 x 10 cells frozen R700 07 Lipofectamine 2000 0 75 mL 11668 027 1 5 mL 11668 019 Opti MEM I Reduced Serum Medium 100 mL 31985 062 500 mL 31985 070 Dulbecco s Modified Eagle Medium 500 mL 11965 092 PE 1000 mL 11965 084 TrypLE Select 1X liquid 500 mL 12563 029 TrypLE Select Animal Origin Free Trypsin Like Enzyme 100 mL 12563 011 Propidium lodide 1 0 mg mL 10 mL P 3566 Blasticidin 50 mg R210 01 Geneticin 20 mL 10131 035 100 mL 10131 027 Fetal Bovine Serum FBS Certified 500 mL 16000 044 Phosphate Buffered Saline PBS pH 7 4 500 mL 10010 023 1L 10010 031 Technical Support Web Resources Visit the Invitrogen website at www invitrogen com for e Technical resources including manuals
18. your lentiviral stocks 2 Transduce the different dilutions of lentivirus in the presence of the polycation Polybrene page 19 3 Determine the Lentiviral titer by fluorescence detection using flow cytometry 2 days after transduction see Important below We do not recommend using fluorescence microscopy to detect EmGFP in your cells from the pLenti7 3 vectors we recommend flow cytometry The pLenti7 3 vectors are designed with EmGFP in their vector backbone which allows for quick screens of transient expression in your cells and titering times of only 2 days While the quantity of cells expressing your gene of interest is significantly greater than other pLenti vectors that do not contain the WPRE and cPPT elements the signal intensity of EmGFP expressed in your cells is not optimal for viewing with fluorescence microscopy For this reason we recommend flow cytometry Continued on next page 21 Titering Your Lentiviral Stock Using EmGFP Continued Materials Needed Trypsin Dissociation Solution Transduction and Titering Procedure for EmGFP 22 Your EmGFP lentiviral stock from the pLenti7 3 V5 TOPO vector or the pLenti7 3 V5 DEST Gateway vector store at 80 C until use Adherent mammalian cell line of choice Complete culture medium for your cell line 96 well tissue culture plates Optional 6 mg mL Polybrene see page 19 Optional TrypLE see page 44 for ordering information trypsin
19. 7 4600 C Ampicillin b a 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 5 821 bp contains the following elements All 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 P 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 P globin polyadenylation signal Allows efficient transcription termination and polyadenylation of mRNA pUC origin of replication ort Permits high copy replication and maintenance in E coli Ampicillin bla resistance gene Allows selection of the plasmid in E coli 43 Additional Products Additional Products 44 Many of the reagents supplied in the ViraPower HiPerform Lentiviral Expression Kits as well as other products suitable for use with the kits are available separately Ordering information for these reagents is provided below For more information go to
20. Carlsbad California 92008 Phone 760 603 7200 or outlicensing lifetech com This product and its use is the subject of U S and foreign patents This product is the subject of U S and foreign patents licensed by Life Technologies Corporation This product is sold for research use only Not for therapeutic or diagnostic use in humans Continued on next page Purchaser Notification Continued Limited Use Label License No 308 WPRE Element This product contains the Woodchuck Post transcriptional Regulatory Element WPRE which is the subject of intellectual property owned by The Salk Institute for Biological Studies and licensed to Life Technologies Corporation The purchase of this product conveys to the buyer the non transferable right to use the purchased amount of the product and components of the product in research conducted by the buyer whether the buyer is an academic or for profit entity The buyer cannot sell or otherwise transfer a this product b its components or c materials made using this product or its components to a third party or otherwise use this product or its components or materials made using this product or its components for Commercial Purposes The buyer may transfer information or materials made through the use of this product to a scientific collaborator provided that such transfer is not for any Commercial Purpose and that such collaborator agrees in writing a not to transfer s
21. Lentiviral Stock Using Blasticidin Continued Transduction and Follow the procedure below to determine the titer of your lentiviral stock Titering Procedure You will use at least one 6 well plate for every lentiviral stock to be titered Blasticidin one mock well plus five dilutions 1 10 11 The day before transduction trypsinize and count the cells and then plate them in a 6 well plate at a density of 2 x 10 cells per well so that they will be 30 50 confluent at the time of transduction Incubate the cells at 37 C overnight in a humidified 5 CO incubator Example When using HT1080 cells plate 2 x 105 cells per well in a 6 well plate On the day of transduction Day 1 thaw your lentiviral stock and prepare 10 fold serial dilutions ranging from 10 to 10 For each dilution dilute the lentiviral stock into complete culture medium to a final volume of 1 mL DO NOT vortex the dilutions Note You may prepare a wider range of serial dilutions 107 to 109 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 see page 19 to each well to a final concentration of 6 pg mL Swirl the plate gently to mix Incubate the plate at 37 C overnight in a humidified 5 CO incubator The following day Day 2 remove the virus containing medium and replace it with 2 mL of complete culture medium Incubate
22. OX4 4GA UK enquiries oxfordbiomedica co uk or BioMedica Inc 11622 EI Camino Real 100 San Diego CA 92130 2049 USA LentiVector is a registered US and European Community trade mark of Oxford BioMedica ple The purchase of this product conveys to the purchaser the limited non transferable right to use the purchased amount of the product only to perform internal research for the sole benefit of the purchaser No right to resell this product or any of its components is conveyed expressly by implication or by estoppel This product is for internal research purposes only and is not for use in commercial services of any kind including without limitation reporting the results of purchaser s activities for a fee or other form of consideration For information on obtaining additional rights please contact outlicensing lifetech com or Out Licensing Life Technologies 5791 Van Allen Way Carlsbad California 92008 The 293FT cell line is genetically modified and carries the pUC derived plasmid pCMVSPORT6TAg neo As a condition of sale this product must be in accordance with all applicable local legislation and guidelines including EC Directive 90 219 EEC on the contained use of genetically modified organisms Gateway Clone Distribution Policy Introduction Gateway Entry Clones Gateway Expression Clones Additional Terms and Conditions The information supplied in this section is intended to provide clarity co
23. V immediate early promoter to control high level expression of the gene of interest in all four vectors e C terminal V5 tag for convenient detection e SV40 promoter driving the expression of Blasticidin pLenti6 3 V5 DEST Gateway vector and pLenti6 3 V5 TOPO Vector or Emerald Green Fluorescent Protein EmGFP derived from Aequorea Victoria GFP pLenti7 3 V5 DEST Gateway and pLenti7 3 V5 TOPO vector e Blasticidin resistance gene Izumi et al 1991 Kimura et al 1994 Takeuchi et al 1958 Yamaguchi et al 1965 for stable transduction and selection in E coli and mammalian cells pLenti6 3 V5 DEST Gateway and pLenti6 3 V5 TOPO TA vectors only or EMGFP pLenti7 3 V5 DEST Gateway and pLenti7 3 V5 TOPO vectors only for easy determination of the Lentiviral titer by flow cytometry Continued on next page System Summary Continued Components of the ViraPower HiPerform Lentiviral Expression System The ViraPower HiPerform Lentiviral Expression Systems facilitate highly efficient in vitro delivery of a target gene to dividing and non dividing mammalian cells using a replication incompetent lentivirus Based on the lentikat system developed by Cell Genesys Dull et al 1998 the ViraPower HiPerform Lentiviral Expression System possesses features that enhance its biosafety while allowing high level gene expression in a wider range of cell types than traditional retrovira
24. ants into cryovials in 1 mL aliquots Store viral stocks at 80 C Proceed to Titering Your Lentiviral Stock page 18 Continued on next page 15 Producing Lentivirus in 293FT Cells Continued Reverse Transfection Procedure Experienced Users 16 If you are an experienced user you may use the reverse transfection procedure to cotransfect 293FT cells For information on positive controls see page 9 Include a negative control no DNA no Lipofectamine 2000 in your experiment to help evaluate results You will need 6 x 10 293FT cells for each sample 1 On Day 1 prepare DNA Lipofectamine 2000 complexes for each transfection sample as follows a Inasterile 5 mL tube dilute 9 ug of the ViraPower Packaging Mix and 3 ug of your pLenti expression plasmid DNA 12 ug total in 1 5 mL of Opti MEM I Medium without serum Mix gently b Ina separate sterile 5 mL tube dilute 36 uL of Lipofectamine 2000 mix gently before use in 1 5 mL of Opti MEM I Medium without serum Mix gently and incubate for 5 minutes at room temperature c After incubation combine the diluted DNA from Step a with the diluted Lipofectamine 2000 from Step b Mix gently d Incubate the tube for 20 minutes at room temperature to allow the DNA Lipofectamine 2000 complexes to form The solution may appear cloudy but this will not impede the transfection 2 While DNA lipid complexes are forming trypsinize and count the 293FT cells R
25. aterials made by the employment of this product or its components for Commercial Purposes The buyer may transfer information or materials made through the employment of this product to a scientific collaborator provided that such transfer is not for any Commercial Purpose and that such collaborator agrees in writing a not to transfer such materials to any third party and b to use such transferred materials and or information solely for research and not for Commercial Purposes Notwithstanding the preceding any buyer who is employed in an academic or government institution may transfer materials made with this product to a third party who has a license from Life Technologies 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 Life Technologies Corporation will not assert a claim against the buyer of infringeme
26. card See Caution previous page Replace the spent medium with 100 uL of fresh growth medium in each well and incubate the plate overnight in a 37 C CO incubator Important Do NOT add Blasticidin to the growth medium After 24 hours incubation Day 3 remove the growth medium from each well and discard Replace the medium in each well with dissociation solution see above Allow the cells to dissociate for 5 minutes at 37 C and then proceed to Preparing Cells for Flow Cytometry next page see Important previous page Continued on next page Titering Your Lentiviral Stock Using EmGFP Continued If you wish to fix your cells before flow cytometry use 2 formaldehyde or Note paraformaldehyde solution in calcium magnesium free PBS Note that these fixatives may increase the autofluorescence of the cells therefore it is critical to include fixed mock transduced cells as a negative control for flow cytometry detection parameters Preparing Cells Prepare your cells for flow cytometry using an FITC filter according to the for Flow protocols established at your flow cytometry facility The steps below provide Cytometry simple guidelines and other methods may be suitable 1 After cell dissociation Steps 6 7 Transduction and Titering Procedure for EmGFP centrifuge cells at 2 000 x g to remove residual media components and then resuspend the cell pellet in a flow cytometry buffer such as calcium magnesium free PBS with
27. ce 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 specific to the epitope tag see your lentiviral vector manual for details Follow the procedure below to transduce the mammalian cell line of choice using the pLenti7 3 vectors 1 Plate the cells in complete medium as appropriate for your application 2 On the day of transduction Day 1 thaw your lentiviral stock and if necessary dilute the appropriate amount of virus see Determining Optimal MOL page 30 into fresh complete medium Keep the total volume of virus containing medium as low as possible to maximize transduction efficiency 3 Remove the culture medium from the cells Mix the virus containing medium gently by pipetting DO NOT vortex and add it to the cells 4 Add Polybrene if desired to the plate at a final concentration of 6 pg mL Swirl the plate gently to mix and incubate it at 37 C in a CO incubator overnight Note If you are transducing cells with undiluted viral stock and are concerned about possible toxicity or growth effects caused by overnight incubation you may incubate the cells for as little as 6 hours before changing the medium 5 The following day Day 2 remove the virus containing medium and replace it with fresh complete culture medium without Blasticidin 6 The following day Day 3 analyze the cells for express
28. concentrations ranging from 2 10 ug mL Blasticidin are sufficient to kill most untransduced mammalian cells Test a range of concentrations see protocol below to ensure that you determine the minimum concentration necessary for your cell line 1 Plate the cells at approximately 25 confluence Prepare a set of 6 7 plates Allow cells to adhere overnight 2 The next day substitute the culture medium with medium containing varying concentrations of Blasticidin 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 that kills the cells within 10 14 days after addition of antibiotic Materials Needed e Your pLenti lentiviral stock from the pLenti6 3 V5 TOPO vector or pLenti6 3 V5 DEST Gateway vector store at 80 C until use e Adherent mammalian cell line of choice e Complete culture medium for your cell line e Optional 6 mg mL Polybrene see page 19 e 6 well tissue culture plates e Crystal violet Sigma Cat no C3886 prepare a 1 crystal violet solution in 10 ethanol e Phosphate Buffered Saline PBS page 44 e Blasticidin 10 mg mL stock as appropriate for selection This component is supplied with the ViraPower HiPerform Lentiviral TOPO Expression TM TM Kit and the ViraPower HiPerform Lentiviral Gateway Expression Kit Continued on next page 26 Titering Your
29. d foreign patents This product is sold under license from Columbia University Rights to use this product are limited to research use only and expressly exclude the right to manufacture use sell or lease this product for use for measuring the level of toxicity for chemical agents and environmental samples in cells and transgenic animals No other rights are conveyed Not for human use or use in diagnostic or therapeutic procedures Inquiry into the availability of a license to broader rights or the use of this product for commercial purposes should be directed to Columbia Innovation Enterprise Columbia University Engineering Terrace Suite 363 New York New York 10027 Continued on next page Purchaser Notification Continued Limited Use Label License No 168 Lanthanide Chelates This product is the subject of U S and foreign patents licensed exclusively to Life Technologies Corporation The purchase of this product conveys to the buyer the non transferable right to use the purchased amount of the product and components of the product in research conducted by the buyer whether the buyer is an academic or for profit entity The buyer cannot sell or otherwise transfer a this product b its components or c materials made using this product or its components to a third party or otherwise use this product or its components or materials made using this product or its components for Commercial Purposes The buyer may transfer infor
30. dini L and Trono D 1998 Self inactivating lentivirus vector for safe and efficient in vivo gene delivery J Virol 72 9873 9880 2010 Life Technologies Corporation All rights reserved The trademarks mentioned herein are the property of Life Technologies Corporation or their respective owners Polybrene is a registered trademark of Abbott Laboratories y 8 For research use only Not intended for any animal or human therapeutic or diagnostic use 55 ll invitrogen by Life technologies Corporate Headquarters 5791 Van Allen Way Carlsbad CA 92008 T 1 760 603 7200 F 1 760 602 6500 E tech supporteinvitrogen com For country specific contact information visit our web site at www invitrogen com
31. e No 108 Lentiviral Technology For additional information about Invitrogen s policy for the use and distribution of Gateway clones see the section entitled Gateway Clone Distribution Policy page 53 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 Blasticidin and the blasticidin resistance gene bsd are the subject of U S patents sold under patent license for research purposes only For information on purchasing a license to this product for purposes other than research contact Licensing Department Life Technologies Corporation 5791 Van Allen Way Carlsbad California 92008 Phone 760 603 7200 Fax 760 602 6500 email outlicensing invitrogen com This product is the subject of U S and foreign patents and is sold under license from the Universit Libre de Bruxelles for research purposes only ccdB selection technology is described in Bernard et al Positive Selection Vectors Using the F Plasmid ccdB Killer Gene Gene 148 1994 71 74 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 For licensing information for use in other than research
32. elow describes the general steps required to express your gene of interest using the ViraPower HiPerform Lentiviral Expression System Refer to the appropriate manual for each pLenti expression vector for instructions to generate your pLenti expression construct 1 Generate the pLenti expression pLenti construct containing your gene Expression of interest Construct ViraPower Packaging Mix 2 Cotransfect the 293FT producer cell line with your pLenti expression construct and the optimized packaging mix 293FT Producer Cell Line 3 Harvest viral supernatant and determine the titer 4 Add the viral supernatant to your mammalian cell line of N interest Select for stably gt transduced cells if desired Your Mammalian Cell Line of Interest promoter gene of interest V5 5 Assay for recombinant protein of interest Methods General Information Introduction Positive Control IM TM The ViraPower HiPerform Lentiviral Expression System is designed to help you create a lentivirus to deliver and express a gene of interest in mammalian cells Although the system has been designed to help you express your recombinant protein of interest in the simplest most direct fashion use of the system is geared towards those 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
33. esuspend the cells at a density of 1 2 x 10 cells mL in growth medium or Opti MEM I Medium containing serum Do not include antibiotics in the medium 3 Add the DNA Lipofectamine 2000 complexes from Step 1d to a 10 cm tissue culture plate containing 5 mL of growth medium or Opti MEM I Medium containing serum Do not include antibiotics in the medium 4 Add 5 mL of the 293FT cell suspension from Step 2 6 x 10 total cells to the plate containing media and DNA Lipofectamine 2000 complexes from Step 3 Mix gently by rocking the plate back and forth Incubate cells overnight at 37 C in a humidified 5 CO incubator 5 The next day Day 2 remove and discard the medium containing the DNA Lipofectamine 2000 complexes and replace it with 10 mL of complete culture medium without antibiotics 6 Incubate the cells for 48 72 hours at 37 C in a humidified 5 CO incubator The difference in viral yield is minimal whether the supernatants are collected 48 or 72 hours after transfection Note Expression of the VSV G glycoprotein causes 293FT cells to fuse resulting in the appearance of large multinucleated cells known as syncytia This morphological change is normal and does not affect production of the lentivirus 7 Posttransfection Day 4 or 5 harvest virus containing supernatants by removing and placing the medium into a 15 mL sterile capped conical tube Caution Remember that you are working with infectious v
34. fold serial dilutions of the lentiviral supernatant 10 to 10 dilutions or untransduced mock following the protocol on page 27 At 48 hours after 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 103 102 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 103 dilution 46 e 10 dilution 5 Thus the titer of this concentrated lentiviral stock is 4 8 x 10 TU mL i e the average of 46 x 10 and 5 x 105 Note that user experience the nature of the gene and vector backbone may affect virus titer e Ifthe titer of your unconcentrated virus is suitable i e 1 x 10 TU mL or higher proceed to Transduction and Analysis page 29 e Ifthe titer of your concentrated lentiviral stock is less than 1 x 10 TU mL produce a new lentiviral stock See Troubleshooting page 33 for more tips and guidelines to optimize your viral yield Transduction and Analysis Introduction Important Transient vs Stable Expression Multiplicity of Infection MOI After you have generated a lentiviral stock with a suitable titer transduce the lentiviral construct into a mammalian cell line of choice and assay for expression of yo
35. for cloning your gene of interest e A corresponding expression control plasmid e One Shot Stbl3 Chemically Competent E coli for transformation Each vector manual supplied with the kit contains a detailed description of the provided reagents and instructions to generate an expression vector with your gene of interest Vector Cat no pLenti6 3 V5 TOPO vector K5310 00 pLenti7 3 V5 TOPO vector K5320 00 pLenti6 3 V5 DEST Gateway vector K5330 00 pLenti7 3 V5 DEST Gateway vector K5340 00 Continued on next page Kit Contents and Storage Continued ViraPower Lentiviral Support Kit Contents The ViraPower HiPerform Lentiviral Support Kit includes the following vectors and reagents Store as directed below Additional packaging mix ViraPower Lentiviral Packaging Mix and Lipofectamine 2000 Reagent may be purchased separately see page 44 for ordering information Important Do not freeze Lipofectamine 2000 Reagent Reagent Composition Amount Storage ViraPower Packaging Mix Contains a mixture of the pLP1 pLP2 and 195 pg 20 C pLP VSVG plasmids 1 ug uL in TE pH 8 0 Lipofectamine 2000 Proprietary 0 75 mL 4 C One Shot StbI3 The following reagents are included with the One Shot Stbl3 Chemically Chemically Competent E coli Genotype of Stbl3 Cells 293FT Cell Line vi Competent E coli kit Transformation efficie
36. h quality goods and services Our goal is to ensure that every customer is 100 satisfied with our products and our service If you should have any questions or concerns about an Invitrogen product or service contact our Technical Support Representatives All Invitrogen products are warranted to 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 the Company s liability to only the price 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 The Company reserves the right to select the method s used to analyze a product unless the Company 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 the Company 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 Support Representatives Life Technologies Corporation shall have no responsibility or liability for any special incidental indirect or consequential loss or damage whatsoever The above limited warranty is sole and exclusive No ot
37. her warranty is made whether expressed or implied including any warranty of merchantability or fitness for a particular purpose 45 Purchaser Notification Limited Use Label License No 19 Gateway Cloning Products 46 This product and its use is the subject of one or more issued and or pending U S and foreign patent applications owned by Life Technologies 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 Life Technologies 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 Life Technologies 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 m
38. invitrogen by technologies ViraPower HiPerform Lentiviral Expression Systems Lentiviral systems for high level expression in dividing and non dividing mammalian cells Cat nos K5310 00 K5320 00 K5330 00 and K5340 00 Rev date 21 December 2010 Manual part no A10290 MAN0000680 ii Contents Kit Contents nd SOTA Co it AD E R AAA da Ai iv MOA Tree 1 SA NN 1 Biosafety Featur s of the System citi oiist niin sn asden krenket n E E EE aes 6 Experimental Matin Goss nica ia eli 8 Ls oe NE 9 General Information e 2 A ER EE R O E tase Uuacsedsp a advan E AN RE 9 Generating Your pLenti Expression Construct rsnsennnrnrnrrnrvrnenvsrsrrrnrvereverevensrsrsrnrnenensvsvsnsavenensvnsrsanesenenenenensnsn 11 Producing Lentivirus in 293FT elsi ian e a ene a e EAE AE E aR EEEE e coin noci n 12 Titering Your Lentiviral Occitan da 18 Titering Your Lentiviral Stock Using EMGFP nsnsenerrrererrvrvrrrrnenevvsrsrrrverererererensnsrsrsrnenensnsrsrnenenenevsnrssravanererenen 21 Titering Your Lentiviral Stock Using Blasio andas 25 Transduction and Analysis si esanen EE A AAA A A WED esd 29 Troubleshooting sa en op asii aiii 33 AAA A e e UE OOO O 37 Bl sticidin iaa a eat 37 E A EN 38 Map and Features of pLP2 nu sirieni en ia enno datado lada dren isbreer 40 Map and Features of pLP VSVG w cccccsceecsssssecsassecnsatasenbecessetecsssssensncsecneatsensnssnansasasnenedancestesnedsessenaaanceateeesaaensnes 42 Additional Produc
39. ion of EmGFP by flow cytometry see Important page 21 7 You may sort the cells expressing EmGFP with flow cytometry and use these cells for assaying protein expression Note Because pLenti7 3 vectors do not contain an antibiotic selection marker they do not generate antibiotic resistant clones Although your gene of interest is integrated into the lentiviral genome there is no antibiotic selection pressure maintaining the integrity of the expression construct As a result depending on the influence of surrounding genomic sequences your construct may change over the course of multiple passages resulting in reduction or loss of protein expression Troubleshooting The table below lists some potential problems and possible solutions that may help you troubleshoot your cotransfection and titering experiments Generating the Lentiviral Stock Observation Reason Solution Low viral titer Low transfection efficiency Used poor quality expression construct plasmid DNA i e plasmid DNA from a mini prep Unhealthy 293FT cells cells exhibit low viability Cells transfected in medium containing antibiotics i e Geneticin Plasmid DNA transfection reagent ratio incorrect Insufficient co transfection 293FT cells plated too sparsely e Do not use mini prep plasmid DNA for transfection Use the PureLink HiPure Plasmid Midiprep Kit or CsCl gradient centrifugation to prepare plasmid DNA e Use healthy 293FT
40. irus at this stage Follow recommended guidelines for working with BL 2 organisms refer to page 7 8 Centrifuge supernatants at 2 000 x g for 15 minutes at 4 C to pellet debris 9 Optional Filter the viral supernatants through a Millex HV 0 45 um or equivalent PVDF filter see Note next page 10 Pipet viral supernatants into cryovials in 1 mL aliquots and store them at 80 C Proceed to Titering Your Lentiviral Stock page 18 Continued on next page Producing Lentivirus in 293FT Cells Continued Note Concentrating Virus Long Term Storage Scaling Up Virus Production It should be possible to use the new ViraPower HiPerform Lentiviral vector constructs for in vivo applications however we have not yet tested the new constructs in vivo If you plan to use your lentiviral construct for in vivo applications filter your viral supernatant through a sterile 0 45 um low protein binding filter after the low speed centrifugation step Step 8 Reverse Transfection Procedure Experienced Users to remove any remaining cellular debris We recommend using Millex HV 0 45 um PVDF filters Millipore Cat no SLHV033RB for filtration If you wish to concentrate your viral stock to obtain a higher titer perform the filtration step prior to concentrating your viral stock It is possible to concentrate VSV G pseudotyped lentiviruses using a variety of methods without significantly affecting their ability to t
41. iviral Stock Continued Preparing and Follow the instructions below to prepare Polybrene Sigma Aldrich Storing Cat no H9268 Polybrene 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 Store stock solutions at 20 C for up to 1 year Do not freeze thaw the stock solution more than 3 times to avoid loss of activity Note The working stock may be stored at 4 C for up to 2 weeks 20 Titering Your Lentiviral Stock Using EmGFP Introduction Experimental Outline Important This section provides guidelines and protocols for titering your lentiviral stock using Emerald Green Fluorescence EmGFP To titer your lentiviral stock using Blasticidin refer to page 25 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 before disposal e Treat used pipettes pipette tips and other tissue culture supplies with bleach and dispose of as biohazardous waste e Wear gloves a laboratory coat and safety glasses or goggles when handling viral stocks and media containing virus To determine the titer of your EmGFP lentiviral stocks you will 1 Prepare a 50 fold or 20 fold serial dilution of
42. ix 9 ug 9 uL of 1 ug uL stock Amount of pLenti expression plasmid 3 pg Amount of Lipofectamine 2000 Reagent 36 pL Note You may produce lentiviral stocks using other tissue culture formats optimization may be necessary to obtain the expected titers If you are producing lentivirus for the first time using the ViraPower System and 293FT cells perform the Forward Transfection procedure on page 15 This procedure requires plating the 293FT cells the day before transfection to obtain cells that are 90 95 confluent Note In previous ViraPower manuals this protocol was referred to as the Alternate Transfection Method If you are an experienced lentivirus user and are familiar with the growth characteristics of 293FT cells you may perform the Reverse Transfection procedure on page 16 In this procedure 293FT cells are added to media containing the DNA Lipofectamine 2000 complexes Continued on next page 13 Producing Lentivirus in 293FT Cells Continued Materials Needed pLenti expression vector containing your gene of interest 0 1 3 0 ug puL in sterile water or TE pH 8 0 e 293FT cells cultured in the appropriate medium i e D MEM containing 10 FBS 2 mM L Glutamine 0 1 mM MEM Non Essential Amino Acids and 1 penicillin streptomycin and 500 pg mL Geneticin Note D MEM already contains 4 mM L Glutamine which is enough to support growth of 293FT cells However since L Glutamine slowly decays over time
43. l systems The System includes the following major components e ApLenti based expression vector into which the gene of interest is cloned The vector contains the WPRE and cPPT elements which allows more cells to express the gene of interest at higher levels and faster titering times The vector also contains the elements required for packaging 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 IM e The ViraPower Packaging Mix containing an optimized mixture of the three packaging plasmids pLP1 pLP2 and pLP VSVG These plasmids supply the helper functions and the structural and replication proteins in trans required to produce the lentivirus The pLP VSVG plasmid in the packaging mix contains the G glycoprotein gene from Vesicular Stomatitis Virus VSV G as a pseudotyping envelope which allows the 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 For more information about the packaging plasmids see the Appendix pages 38 43 e An optimized 293FT producer cell line that stably expresses the SV40 large T antigen under the control of the human CMV promoter and facilitates optimal production of virus For more information about the 293FT Cell Line refer to the 293FT Cell Line manual TM Y
44. ll 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 MOT increases Continued on next page 29 Transduction and Analysis Determining the Optimal MOI l RECO 7 tr S N Positive Control Note 30 A number of factors influence optimal MOI including e The nature of your mammalian cell line e g non dividing vs dividing cell type see Recommendation below e The transduction efficiency of your mammalian cell line e The nature of your target gene of interest e Your application of interest If you are transducing the lentiviral construct into your mammalian cell line for the first time use a range of MOTs e g 0 0 5 1 5 10 to determine the MOI required to obtain the optimal protein expression for your application We have found that in general 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 Cont
45. ll the DNA Lipofectamine 2000 complexes dropwise to the plate of 293FT cells Steps 1 and 2 Mix gently by rocking the plate back and forth Incubate the cells overnight at 37 C in a humidified 5 CO incubator 5 The next day Day 3 remove the cell culture plate containing the 293FT cells with DNA Lipofectamine complexes from the incubator Remove and discard the medium containing the DNA Lipofectamine 2000 complexes and replace it with 10 mL of complete culture medium without antibiotics 6 Incubate the cells for 48 72 hours at 37 C in a humidified 5 CO incubator Collecting the supernatant at 48 or 72 hours post transfection makes minimal difference in viral yield 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 lentivirus production 7 Post transfection Day 5 or 6 harvest virus containing supernatants by removing and transferring the medium into a 15 mL sterile capped conical tube Caution Remember that you are working with infectious virus at this stage Follow recommended guidelines for working with BL 2 organisms refer to page 7 8 Centrifuge the supernatants at 2 000 x g for 15 minutes at 4 C to pellet debris 9 Optional Filter the viral supernatants through a Millex HV 0 45 um or an equivalent PVDF filter see Note page 17 10 Pipet viral supernat
46. mation 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 Life Technologies 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 Life Technologies is willing to accept return of the product with a full refund For information on purchasing a license to this product fo
47. ncerning Invitrogen s policy for the use and distribution of cloned nucleic acid fragments including open reading frames created using Invitrogen s commercially available Gateway Technology Invitrogen understands that Gateway entry clones containing attL1 and attL2 sites may be generated by academic and government researchers for the purpose of scientific research Invitrogen agrees that such clones may be distributed for scientific research by non profit organizations and by for profit organizations without royalty payment to Invitrogen Invitrogen also understands that Gateway expression clones containing attB1 and attB2 sites may be generated by academic and government researchers for the purpose of scientific research Invitrogen agrees that such clones may 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
48. ncy is gt 1 x 10 cfu ug plasmid DNA Store at 80 C Amount 6 mL Reagent Composition S O C Medium 2 Tryptone 0 5 Yeast Extract 10 mM NaCl 25mMKCI 10 mM MgCl 10 mM MgSO 20 mM glucose Stb13 Cells pUC19 Control DNA 10 pg pL in 5 mM Tris HCl 0 5 mM EDTA pH 8 0 21 x 50 pL 50 pL F mcrB mrr hsdS20 rs my recA13 supE44 ara 14 galK2 lacY1 proA2 rpsL20 Str xyl 5 A leu mtl 1 Note This strain is endA1 TM TM Each ViraPower HiPerform Lentiviral Expression Kit includes the 293FT producer cell line The 293FT Cell Line is supplied as one vial containing 3 x 10 frozen cells in 1 mL of Freezing Medium Upon receipt store in liquid nitrogen For instructions on how to thaw culture and maintain the 293FT Cell Line see the 293FT Cell Line manual included with the ViraPower HiPerform Lentiviral Expression Kit To download the manual visit www invitrogen com or contact Technical Support page 45 Introduction System Summary Description of the System ViraPower HiPerform Lentiviral Expression Vectors TM TM The new ViraPower HiPerform Lentiviral Expression Systems allow the creation of a replication incompetent HIV 1 based lentivirus to deliver and express a gene of interest in dividing or non dividing mammalian cells The new expression systems use four new expression vectors e pLenti6 3 V5 DEST Gateway
49. nt of the above patents based upon the manufacture use or sale of a therapeutic clinical diagnostic vaccine or prophylactic product developed in research by the buyer in which this product or its components was employed provided that none of i this product ii any of its components or iii a method claim of the foregoing patents was used in the manufacture of such product Life Technologies Corporation will not assert a claim against the buyer of infringement of patents owned or controlled by Life Technologies 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 Life Technologies 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 Life Technologies Corporation 5791 Van Allen Way Carlsbad California 92008 Phone 760 603 7200 email outlicensing invitrogen com Continued on next page Purchaser Notification Continued Gateway Clone Distribution Policy Limited Use Label License No 27 RNA Transfection Limited Use Label License No 51 Blasticidin and the Blasticidin Selection Marker Limited Use Label License No 54 ULB ccdB Selection Technology Limited Use Label Licens
50. ock continued Observation Reason Solution Low viral titer 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 19 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 17 Inserts larger than 5 6 kb are not recommended Polybrene not included during titering procedure Transduce the lentiviral construct into cells in the presence of Polybrene Lipofectamine 2000 handled incorrectly e Store at 4 C Do not freeze e Mix gently by inversion Do not vortex Using fluorescence microscopy to view EmGFP titer The signal level of EmGFP in the cells is not optimal for visual evaluation using fluorescence microscopy We recommend using only flow cytometry to evaluate transduction efficiency No colonies obtained upon titering Too much antibiotic used for selection Determine the antibiotic sensitivity of your cell line by performing a kill curve experiment Use the minimum amount of antibiotic required to kill your untransduced cell line Viral stocks stored incorrectly Aliquot and store stocks at 80 C Do not freeze thaw more than 3 times Polybrene not included during transduction Tran
51. ons and transduced into a mammalian cell line available separately see page 44 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 Continued on next page System Summary Continued Advantages of the System How Lentivirus Works TM TM Using the ViraPower HiPerform Lentiviral Expression System to facilitate lentiviral based expression of the gene of interest provides the following advantages Offers you a choice to use Gateway technology Cat nos K5330 00 and K5340 00 or TOPO Cloning technologies Cat nos K5310 00 and K5320 00 Promotes enhanced protein expression up to 4 fold or greater Generates an HIV 1 based lentivirus that effectively transduces dividing and non dividing mammalian cells thus broadening the potential applications beyond those of traditional Moloney Murine Leukemia Virus MoMLV based retroviral systems Naldini 1998 Efficiently delivers the gene of interest to mammalian cells in culture or in vivo Dull et al 1998 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 Produces a pseudotyped virus with a broadened host range Yee et al 1994 Includes multiple features designed to enhance the biosafety of the system pLenti6 3 series vectors offe
52. other than research contact Licensing Department Life Technologies Corporation 5791 Van Allen Way Carlsbad California 92008 Phone 760 603 7200 Fax 760 602 6500 or The Salk Institute for Biological Studies 10010 North Torrey Pines Road La Jolla CA 92037 Attn Office of Technology Management Phone 858 453 4100 extension 1275 Fax 858 546 8093 Continued on next page 51 Purchaser Notification Continued Limited Use Label License No 317 LentiVector Technology Limited Use Label License No 358 Research Use Only Information for European Customers 52 This product is licensed under U S and foreign patents from Oxford BioMedica UK Ltd Oxford UK and is provided for use in academic and commercial in vitro and in vivo research for elucidating gene function and for validating potential gene products and pathways for drug discovery and development but excludes any use of LentiVector technology for creating transgenic birds for the purpose of producing useful or valuable proteins in the eggs of such transgenic birds the delivery of gene therapies and for commercial production of therapeutic diagnostic or other commercial products not intended for research use where such products do not consist of or incorporate a lentiviral vector Information about licenses for commercial uses excluded under this license is available from Oxford BioMedica UK Ltd Medawar Centre Oxford Science Park Oxford
53. ou will cotransfect the ViraPower Packaging Mix and the pLenti vector containing the gene of interest into 293FT cells to produce a replication incompetent lentivirus and then use this lentivirus to transduce a mammalian cell line of interest Continued on next page System Summary Continued Features of the ViraPower HiPerform Lentiviral Systems TM TM The major features of the ViraPower HiPerform Lentiviral Systems include An expression plasmid containing the gene of interest under the control of a CMV early promoter and elements that allow packaging of the construct into virions Polypurine Tract from HIV cPPT for increased viral titer Park et al 2001 WPRE for increased transgene expression Zufferey et al 1999 An optimized mix of the three packaging plasmids pLP1 pLP2 and pLP VSVG that supply the structural and replication proteins in trans that are required to produce the lentivirus The 293FT cell line which allows the production of lentivirus following cotransfection of the expression plasmid and the plasmids in the packaging mix Control expression plasmid to optimize virus production and cell transduction containing either The lacZ gene which when packaged into virions and transduced into a mammalian cell line expresses P galactosidase included with each expression vector kit or The Emerald Green Fluorescent Protein EmGFP gene which expresses EmGFP when packaged into viri
54. ou wish to control the number of integrated copies of the lentivirus e You wish to generate reproducible expression results This section provides guidelines and protocols for titering your lentiviral stock In addition to higher expression of the gene of interest all ViraPower HiPerform Lentiviral vectors yield a higher Blasticidin Bsd or Emerald Green Fluorescence EmGFP titer compared to previous pLenti vectors The pLenti6 3 vectors K5310 00 and K5330 00 contain Bsd in the vector backbone which allows titer of active virus by selection of Blasticidin resistant clones after transduction Alternatively pLenti7 3 FastTiter vectors K5320 00 and K5340 00 contain the EmGFP reporter gene in the vector backbone which allows titer by flow cytometry in only 2 days post transduction For Titering lentiviral stock using EmGFP refer to page 21 For Titering lentiviral stock using Blasticidin refer to page 25 IM TM TM ViraPower HiPerform Lentiviral FastTiter Expression kits K5320 00 and K5340 00 allow you to titer lentivirus in only 2 days because the pLenti7 3 vectors contain EMGFP reporter gene in the vector backbone instead of Bsd This feature makes these kits ideal for high throughput and quick screens of transient expression using flow cytometry TM Important The FastTiter Expression kits are optimal for quick screens of transient expressions using flow cytometry The signal intensity produced by the
55. our 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 to 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 Continued on next page 35 Troubleshooting Continued Transducing Mammalian Cells continued Observation Reason Solution Cytotoxic effects Large volume of viral e Remove the spent media containing observed after supernatant used for virus and replace with fresh complete transduction transduction media e Concentrate the virus Yee 1999 Polybrene used during transduction Verify the sensitivity of your cells to Polybrene If cells are sensitive omit the Polybrene during transduction Too much antibiotic used for selection Determine the antibiotic sensitivity of your cell line by performing a kill curve Use the minimum concentration of antibiotic required to kill your untransduced cell line
56. pLenti7 3 V5 DEST Gateway Vector Kit v ViraPower Lentiviral Support Kit Y Y Y Y One Shot Stb13 Chemically Competent E coli Y v v v 293FT Cell Line v v Y Y Blasticidin Y Y Continued on next page Kit Contents and Storage Continued Shipping and The ViraPower HiPerform Lentiviral products are shipped as described Storage below Upon receipt store each component as detailed below Item Shipping Storage 293FT Cell Line Dry ice Liquid nitrogen Blasticidin Room temperature 20 C ViraPower Packaging Mix Room temperature 20 C ViraPower Lentiviral Support Kit Blue ice e ViraPower Packaging Mix 20 C e Lipofectamine 2000 4 C do not freeze pLenti6 3 V5 TOPO TA Cloning Kit Dry ice e Vectors 20 C e One Shot Stbl3 Chemically Competent E coli 80 C pLenti7 3 V5 TOPO TA Cloning Kit Dry ice e Vectors 20 C e One Shot Stbl3 Chemically Competent E coli 80 C pLenti6 3 V5 DEST Gateway Vector Kit Dry ice e Vectors 20 C e One Shot Stbl3 Chemically Competent E coli 80 C pLenti7 3 V5 DEST Gateway Vector Kit Dry ice e Vectors 20 C e One Shot Stbl3 Chemically Competent E coli 80 C Expression Vectors TM TM Each ViraPower HiPerform Lentiviral Expression Kit also includes a pLenti based expression vector kit The expression vector kit includes e A pLenti based expression vector
57. please contact Out Licensing Life Technologies Corporation 5791 Van Allen Way Carlsbad California 92008 Phone 760 603 7200 or e mail TM The Lentiviral Technology based upon the lentikat system is licensed from Cell Genesys Inc under U S 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 whether the buyer is an academic or for profit entity including non gene therapy research and target validation applications in laboratory animals Continued on next page 47 Purchaser Notification Continued Limited Use Label License No 109 Retroviral Helper Lines Limited Use Label License No 127 GFP with Heterologous Promoter 48 Retroviral helper cell lines are licensed from Wisconsin Alumni Research Foundation under U S Patents 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 Life Technologies This product and its use is the subject of U S an
58. ppropriately sized tissue culture plates for your application e Blasticidin as appropriate if selecting for stably transduced cells pLenti6 3 vectors only Blasticidin is supplied with the ViraPower HiPerform Lentiviral TOPO Expression Kit and the ViraPower HiPerform Lentiviral Gateway Expression Kit TM Follow the procedure below to transduce the mammalian cell line of choice using the pLenti6 3 vectors 1 Plate the cells in complete medium as appropriate for your application 2 On the day of transduction Day 1 thaw your lentiviral stock and if necessary dilute the appropriate amount of virus into fresh complete medium to obtain a suitable MOI Keep the total volume of the virus containing medium as low as possible to maximize transduction efficiency Do not vortex 3 Remove the culture medium from the cells Mix the virus containing medium gently by pipetting and add it to the cells 4 Add Polybrene if desired to a final concentration up to 10 ug mL Swirl the plate gently to mix and incubate it 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 you may incubate the cells for as little as 6 hours before changing the medium 5 The following day Day 2 remove the virus containing medium and replace it with fresh complete culture medium Incuba
59. r purposes other than research contact Out Licensing Life Technologies 5791 Van Allen Way Carlsbad California 92008 Phone 760 603 7200 Fax 760 602 6500 E mail outlicensing lifetech com Continued on next page 49 Purchaser Notification Continued Limited Use Label License No 198 Fluorescent proteins and stable cell lines expressing such proteins but not for vectors that contain the genes for such fluorescent proteins Limited Use Label License No 267 Mutant GFP Products Limited Use Label License No 272 Humanized GFP 50 This product and its use is the subject of U S and foreign patents The purchase of this product conveys to the buyer the nontransferable 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 No rights are conveyed to modify or clone the gene encoding GFP contained in this product 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 Commercial P
60. r significantly improved levels of protein expression by increasing the number of cells that express the cloned gene of interest pLenti7 3 series vectors offer significantly improved levels of expression of your gene of interest pLenti7 3 vectors also allow high speed and high throughput titering applications using EMGFP and reduce the titering time to 2 days When 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 Continued on next page System Summary Continued TM TM Purpose of this This manual presents an overview of the ViraPower HiPerform Lentiviral Manual Expression System and provides instructions and guidelines to M 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 a mammalian cell line of choice Assay for transient expression of your recombinant protein or Generate a stably transduced cell line For details and instruction
61. ransduce cells If your cell transduction experiment requires a relatively high Multiplicity of Infection MOI concentrate your virus before titering and proceeding to transduction For details and guidelines to concentrate your virus supernatant by ultracentrifugation refer to published reference sources Yee 1999 Place lentiviral stocks at 80 C for long term storage Repeated freezing and thawing may result in loss of viral titer and is not recommended When stored properly viral stocks of an appropriate titer are suitable for use for up to one year After long term storage re titer viral stocks before transducing your mammalian cell line of interest You may scale up the cotransfection experiment to produce a larger volume of lentivirus 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 To scale up your cotransfection 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 17 Titering Your Lentiviral Stock Introduction ViraPower HiPerform Lentiviral FastTiter Expression Kits 18 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 Y
62. ransfections in 10 cm plates Note ViraPower Packaging Mix is available separately or as part of the ViraPower Lentiviral Support Kits page 44 TM TM The human 293FT Cell Line supplied with the ViraPower HiPerform Lentiviral Expression kits facilitates 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 page 44 For more information about pCMVSPORT6TAg neo and how to culture and maintain 293FT cells refer to the 293FT Cell Line manual This manual is supplied with the ViraPower HiPerform Lentiviral Expression kits and is also available at www invitrogen com or by contacting Technical Support page 45 Note The 293FT Cell Line is also available separately see page 44 The health of your 293FT cells at the time of transfection is critical to the success of lentivirus production Use of unhealthy cells can 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 cells are greater than 90 viable e Subculture and maintain cells in complete medium containing 0 1 mM MEM
63. re 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 P 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 P 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 39 Map and Features of pLP2 pLP2 Map The figure below shows the features of the pLP2 vector The sequence of pLP2 is available at www invitrogen com or by contacting Technical Support see page 45 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
64. reduced to three i e gag pol and rev e 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 e 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 e Although the three packaging plasmids allow in trans expression 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 actually present in the packaged viral genome and thus are never expressed in the transduced target cell No new replication competent virus can be produced e The lentiviral particles produced in this system are replication incompetent and only carry the gene of interest No other viral species are produced e 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 e A constitutive promoter RSV promoter has been placed upstream of the 5 LTR in the pLenti e
65. rol lentiviral vectors expressing lacZ are available for optimization see your vector manual and page 44 for information If you have generated a lentiviral stock of a lacZ expression control pLenti6 3 V5 GW lacZ or pLenti6 3 V5 GW EmGFP use the stock to help you determine the optimal MOI for your particular cell line and application After you have transduced the control lentivirus into a mammalian cell line the gene encoding B galactosidase is constitutively expressed and can be easily assayed refer to the expression vector or expression control vector manual for assay methods Viral supernatants are generated by harvesting spent media containing virus from the 293FT producer cells Spent media lacks nutrients and may contain some toxic metabolic waste products If you are using a large volume of viral supernatant to transduce your mammalian cell line e g 1 mL of viral supernatant per well in a 6 well plate the 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 Continued on next page Transduction and Analysis Continued Materials Needed Transduction Procedure for Blasticidin e Your titered lentiviral stock page 18 store at 80 C until use e Mammalian cell line of choice e Complete culture medium for your cell line e 6mg mL Polybrene if desired page 19 e A
66. s to generate your expression vector refer to the manual for the pLenti vector you are using For instructions to culture and maintain the 293FT producer cell line refer to the 293FT Cell Line manual These manuals are supplied with the ViraPower HiPerform Lentiviral Expression Kits and are also available at www invitrogen com or by contacting Technical Support page 45 Biosafety Features of the System Introduction The ViraPower HiPerform Lentiviral Expression System is a fourth generation lentiviral vector based system developed by Dull et al 1998 It includes a significant number of safety features designed to enhance its biosafety and to minimize its relation to the wild type human HIV 1 virus These safety features are discussed below TM TM Biosafety Features The ViraPower HiPerform Lentiviral Expression System includes the of the ViraPower following key safety features HiPerform e The pLenti expression vector contains a deletion in the 3 LTR AU3 that Lentiviral System does not affect the 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 e The number of genes from HIV 1 used in the system has been
67. sduce the lentiviral construct into cells in the presence of Polybrene Titer indeterminable cells confluent Too little antibiotic used for selection Increase amount of antibiotic Viral supernatant not diluted sufficiently Titer lentivirus using a wider range of 10 fold serial dilutions e g 10 to 10 34 Continued on next page Troubleshooting Continued The table below lists some potential problems and possible solutions that may help you troubleshoot your transduction and expression experiment Transducing Mammalian Cells Observation Reason Solution No expression of the gene of interest Promoter silencing 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 Viral stocks stored incorrectly Aliquot and store stocks at 80 C Do not freeze thaw more than 3 times Frozen cells used for expression experiments for pLenti7 3 vector pLenti7 3 vectors are designed for transient expression We do not recommend using frozen cells for these expression experiments Poor expression of the gene of interest Low transduction efficiency 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 y
68. se kits is not optimal for detection using fluorescence microscopy We recommend flow cytometry to detect the EmGFP in your transduced cells Continued on next page Titering Your Lentiviral Stock Continued Factors Affecting Viral Titer Selecting a Cell Line for Titering Using Polybrene During Transduction e A number of factors can influence viral titers including e The size of your gene of interest Titers decrease as the size of the insert increases We have determined that virus titer drops approximately 2 fold for each kb over 4 kb of insert size To produce a lentivirus with an insert of gt 4 kb concentrate the virus to obtain a suitable titer see page 17 The size of the wild type HIV genome is approximately 10 kb Because the size of the elements required for expression from pLenti vectors total approximately 4 4 4 kb the size of your insert should not exceed 5 6 kb e The characteristics of the cell line used for titering We strongly recommend the human fibrosarcoma line HT1080 as the gold standard for reproducibly titering lentivirus However other cell lines may also be used In general these cells should belong to an adherent non migratory cell line and exhibit a doubling time in the range of 18 25 hours e The age of your lentiviral stock Viral titers may decrease with long term gt 1 year storage at 80 C If your lentiviral stock has been stored for longer than 6 months titer your len
69. te the cells at 37 C in a humidified 5 CO incubator overnight 6 The following day Day 3 perform one of the following and then proceed to Step 7 e If you are performing transient expression experiments harvest the cells and assay for expression of your recombinant protein or e To select for stably transduced cells remove the spent medium and replace it with fresh complete medium containing the appropriate amount of Blasticidin 7 Replace the spent medium with fresh medium containing Blasticidin every 34 days until antibiotic resistant colonies can be identified generally 10 to 12 days after selection 8 Pick at least 5 antibiotic resistant colonies see the Note on page 32 and expand each clone to assay for expression of the recombinant protein Continued on next page 31 Transduction and Analysis Continued Note Detecting Recombinant Protein Transduction Procedure for EmGFP 32 Integration of the lentivirus into the genome is random Depending upon the influence of the surrounding genomic sequences at the integration site you may observe varying levels of recombinant protein expression from different antibiotic resistant clones Test at least 5 antibiotic resistant clones and select the clone that provides the optimal expression of your recombinant protein for further studies You may use any method to detect your recombinant protein of interest including functional analysis immunofluorescen
70. tiviral stock prior to use e Number of freeze thaw cycles Viral titers can decrease as much as 10 with each freeze thaw cycle e Improper storage of your lentiviral stock Lentiviral stocks should be stored at 80 C in cryovials We strongly recommend the human fibrosarcoma line HT1080 ATCC Cat no CCL 121 as the gold standard for reproducibly titering lentivirus However you may use the same mammalian cell line that you use for your expression studies to titer your lentiviral stocks 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 titer all of the lentiviral constructs using the same mammalian cell line For more information on cells used for titering see Factors Affecting Viral Titer above Lentivirus transduction may be enhanced if cells are transduced in the presence of hexadimethrine bromide Polybrene Sigma Aldrich Cat no H9268 For best results we recommend performing transduction in the presence of Polybrene Note however that some cells are sensitive to Polybrene e g primary neurons Before performing any transduction experiments test your cell line for sensitivity to Polybrene at a range of 0 to 10 ug mL If your cells are sensitive to Polybrene e g exhibit toxicity or phenotypic changes do not add Polybrene during transduction Continued on next page 19 Titering Your Lent
71. ts arica aaa dia SER 44 Technical SUpport sen eene E E ae E AE AEE Ad E EEE A RET tage 45 Purchaser Notification ci ta 46 Gateway Clon DS A NE 53 REFERENCES O NN RAN 54 iii Kit Contents and Storage Types of Kits This manual is supplied with the kits listed below The ViraPower HiPerform Lentiviral Expression Kits include the ViraPower HiPerform Lentiviral Support Kit an expression vector and the 293FT producer cell line The ViraPower Lentiviral Support Kits include the ViraPower Packaging Mix Lipofectamine 2000 and a selection agent Product Cat no ViraPower HiPerform Lentiviral TOPO Expression Kit K5310 00 ViraPower HiPerform Lentiviral FastTiter TOPO Expression Kit K5320 00 ViraPower HiPerform Lentiviral Gateway Expression Kit K5330 00 ViraPower HiPerform Lentiviral FastTiter Gateway Expression Kit K5340 00 Intended Use For Research Use Only Not intended for any animal or human therapeutic or diagnostic use System The following table shows the components associated with ViraPower Components HiPerform Lentiviral Expression Kits For detailed instructions to grow and maintain the 293FT Cell Line see the 293FT Cell Line manual Cat no Components K5310 00 K5320 00 K5330 00 K5340 00 pLenti6 3 V5 TOPO TA Cloning Kit Y pLenti7 3 V5 TOPO TA Cloning Kit Y pLenti6 3 V5 DEST Gateway Vector Kit v
72. ty glasses or goggles when handling viral stocks and media containing virus Experimental To determine the titer of your lentiviral stocks using Blasticidin you will Outline 1 Prepare 10 fold serial dilutions of your lentiviral stocks 2 Transduce the different dilutions of lentivirus in the presence of the polycation Polybrene into a mammalian cell line HT1080 is recommended 3 Select for stably transduced cells using Blasticidin 4 Stain and count the number of Blasticidin resistant colonies in each dilution Antibiotic The pLenti6 3 expression constructs contain the Blasticidin resistance gene bsd Selection Kimura et al 1994 to allow for Blasticidin selection Takeuchi et al 1958 Yamaguchi et al 1965 of mammalian cells that have stably transduced the lentiviral construct TM TM Blasticidin is supplied with the ViraPower HiPerform Lentiviral Expression Kit and is also available separately See page 44 for ordering information Preparing For more information about how to prepare and handle Blasticidin refer to the Blasticidin Appendix page 37 Continued on next page 25 Titering Your Lentiviral Stock Using Blasticidin Continued Determining To select for stably transduced cells using Blasticidin you must first determine Antibiotic the minimum concentration of Blasticidin required to kill your untransduced Sensitivity mammalian cells i e perform a kill curve experiment Typically
73. uch 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 and or 4 resale of the product or its components whether or not such product or its components are resold for use in research In addition any use of WPRE outside of this product or the product s authorized use requires a separate license from the Salk Institute Life Technologies will not assert a claim against the buyer of infringement of patents owned by Life Technologies and claiming this product based upon the manufacture use or sale of a therapeutic clinical diagnostic vaccine or prophylactic product developed in research by the buyer in which this product or its components was employed provided that neither this product nor any of its components was used in the manufacture of such product or for a Commercial Purpose If the purchaser is not willing to accept the limitations of this limited use statement Life Technologies is willing to accept return of the product with a full refund For information on purchasing a license to this product for purposes
74. ur 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 it is transduced into mammalian cells Once integrated into the genome the lentivirus can no longer produce packageable virus After transducing your lentiviral construct into a mammalian cell line you may assay for the expression of your gene of interest in the following ways e For pLenti6 3 and pLenti 7 3 vectors pool a heterogeneous population of cells and test for expression directly after transduction i e transient expression Note that you must wait for a minimum of 48 72 hours after transduction before harvesting your cells to allow the transduced cells to accumulate the expressed protein e For pLenti6 3 vectors only select for stably transduced cells using Blasticidin This requires a minimum of 10 12 days after transduction but allows the generation of clonal cell lines that stably express the gene of interest Be aware that the pLenti7 3 vectors are used for transient expression only and do not produce stably transduced cells Note We have observed stable expression of a target gene for at least 6 weeks following transduction and selection To obtain optimal expression of your gene of interest transduce the lentiviral construct into a mammalian cell line of choice using a suitable MOL MOI is defined as the number of virus particles per ce
75. urnal of Antibiotics Series A 11 1 5 White S M Renda M Nam N Y Klimatcheva E Y Zhu Fisk J Halterman M Rimel B J Federoff H Pandya 5 Rosenblatt J D and Planelles V 1999 Lentivirus vectors using human and simian immunodeficiency virus elements J Virology 73 2832 2840 Yamaguchi H Yamamoto C and Tanaka N 1965 Inhibition of Protein Synthesis by Blasticidin S I Studies with Cell free Systems from Bacterial and Mammalian Cells J Biochem Tokyo 57 667 677 Yee J K Miyanohara A LaPorte P Bouic K Burns J C and Friedmann T 1994 A General Method for the Generation of High Titer Pantropic Retroviral Vectors Highly Efficient Infection of Primary Hepatocytes Proc Natl Acad Sci USA 91 9564 9568 Yee J K 1999 in The Development 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 Nal
76. urpose 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 Life Technologies 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 this product or 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 Life Technologies Corporation 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 Life Technologies Corporation 5791 Van Allen Way
77. vector maps and sequences application notes SDSs FAQs formulations citations handbooks etc e Complete technical support contact information e Access to the Invitrogen Online Catalog e Additional product information and special offers Contact Us For more information or technical assistance call write fax or email Additional international offices are listed on our website www invitrogen com Corporate Headquarters Japanese Headquarters European Headquarters 5791 Van Allen Way 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 supportQinvitrogen com E mail jpinfoQinvitrogen com E mail eurotech invitrogen com SDS Certificate of Analysis Limited Warranty Safety Data Sheets SDSs are available on our website at www invitrogen com sds The Certificate of Analysis provides detailed quality control and product qualification information for each product Certificates of Analysis are available on our website Go to www invitrogen com support and search for the Certificate of Analysis by product lot number which is printed on the box Invitrogen a part of Life Technologies Corporation is committed to providing our customers with hig
78. vides the highest transfection efficiency in 293FT cells e DNA Lipofectamine 2000 complexes can be added directly to cells in culture medium in the presence of serum e Removal of complexes or medium change or addition following transfection are not required although complexes can be removed after 4 to 6 hours without loss of activity Note Lipofectamine 2000 is available separately or as part of the ViraPower HiPerform Lentiviral Support Kits see page 44 To facilitate optimal formation of DNA Lipofectamine 2000 complexes we recommend using Opti MEM I Reduced Serum Medium see page 44 Generating Your pLenti Expression Construct Introduction DNA Isolation Guidelines Important To generate a pLenti expression construct containing your gene of interest refer to your specific vector s manual for instructions After you have created your expression construct isolate plasmid DNA for transfection Important You should verify that your lentiviral plasmid has not undergone aberrant recombination by performing an appropriate restriction enzyme digest See the vector manual for details Plasmid DNA for transfection into eukaryotic cells must be clean and free from contamination with phenol and sodium chloride contamination Contaminants may kill the cells and salt interferes with lipid complexing decreasing transfection efficiency When using commercially available kits to isolate plasmid DNA from E
79. virus Dilution EmGFP Positive Cells 107 91 5 10 3 34 6 10 4 4 In the above example the 10 dilution is used to calculate the titer because the percentage of EmGFP positive cells falls into the desired range of 1 30 The frequency of EmGFP positive cells is 4 4 100 0 044 multiplied by 2 x 10 the number of cells in the well divided by 1 the volume of inoculum Thus titer in this example is 0 044 x 200 000 1 x 10 8 8 x 107 TU mL We typically obtain unconcentrated EmGFP lentivirus titers in the range of 5 x 10 2 x 10 TU mL To obtain higher lentivirus titers concentrate your virus see page 17 The titer of concentrated lentivirus stocks may be up to 1 x 10 TU mL Titering Your Lentiviral Stock Using Blasticidin Introduction This section provides guidelines and protocols for titering your lentiviral stock using Blasticidin see page 44 for ordering information To titer your lentiviral stock using EmGFP refer to page 21 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 before disposal 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 safe
80. xpression 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 because 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 Exercise extra caution when creating lentivirus that carry potential harmful or toxic genes e g activated oncogenes For more information about the BL 2 guidelines and lentivirus handling refer to the document Biosafety in Microbiological and Biomedical Laboratories 5 Edition published by the Centers for Disease Control CDC This document is available at www cdc gov biosafety publications index htm Handle all lentiviruses in compliance with established institutional guidelines Because safety requirements for use and handling of lentiviruses may vary at individual institutions consult the health and safety guidelines and or officers at your institution before using the ViraPower HiPerform Lentiviral Expression System Experimental Outline Flow Chart The diagram b

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