Lenti-III-HA Tag Expression Systems
Contents
1. VSV Glycoprotein Envelope Most commercial retroviral vectors are limited in gene delivery ap plications because of their restricted tropisms and generally low titres For re combinant lentiviral vectors these limitations are resolved by pseudotyping the vector with the G glycoprotein gene from Vesicular Stomatitis Virus Gly coprotein VSV G envelope The significant advantages associated with the use of VSV G envelope include allowing production of high titre lentiviral vectors increasing viral particle stability broadening target cell ranges generating highly efficient transduction efficiency Burns et al 1993 Emi et al 1991 Yee et al 1987 1994 1999 Packaging Limits E Recombinant lentiviral titres will decrease with increasing insert gene size The packaging limit for our Lenti IlI HA Tag expression system is approxi mately 5 5 kb above these limits little to no virus will be produced Lenti Ill HA Tag Handbook Page 6 of 16 T x us zm B C o D 5 mE AE an gt a te Materials Scientists at abm have successfully developed a comprehensive product line for HA tagged gene expression and reagents for packaging viral particles In addition ready to use lentiviral particles are also available for immediate transduction of any target cells as in shown in Table I Table Lenti Ill HA Tag Vector and Kits Kit Cat No Component Cat No Quantity2. 6273 3022 Email allan bio rev com bio rev com United Kingdom NBS Biologicals Ltd Tel 44 0 1480 433875 Email info nbsbio co uk www nbsbio co uk Page 16 of 16 The Source for any Antibody siRNA and Viral Vector The Depot for PCR qPCR and Transfection Reagents
3. Combo Mix LV0O3 sup plies all the structural and replication proteins in trans that are re quired for high titre lentivirus production in packaging cells Titres can be obtained up to 107IU ml Figure 2 on page 4 shows the vector map for the pLentiHlll HA vector used in this kit Page 5 of 16 Lenti IlI HA Tag Handbook Lenti Ill HA Tag Expression System General Information about Lentiviral Vectors Morphology Virions consist of an envelope a nucleocapsid a nucleoid and matrix proteins The enveloped virions assume a spherical to pleomor phic shape of 80 100nm in diameter The virion surface is covered with dense inconspicuous spikes of 8 nm in length Physical Properties Virions have a buoyant density of 1 13 1 18g cm in su crose Virions are sensitive to treatment with heat detergents and formalde hyde The infectivity is not affected by irradiation How Lentiviruses Work Once target cells are transduced with a recombinant lentivirus the viral RNA is reverse transcribed actively imported into the nucleus Lewis amp Emerman 1994 Naldini 1999 and undergoes stable integration into the host genome Buchschacher amp Wong Staal 2000 Luciw 1996 One or two days after the lentiviral genome is integrated into the host genome you may begin to assay for the transient expression of your recombinant protein or use ap propriate selection markers to generate a stable cellline for long term expres sion studies
4. G327 G328 LV098 LV099 pLenti II HA Vector LVO22 10ug i i Packaging Mix LV003 100u vi v J Lentifectin G074 1 0ml vl y 293T Cells LVO10 xO E vi Lenti GFP Vector LVO11 a 10ug J i Additional Materials Required The following materials and reagents are required but not provided Dulbecco s Modified Eagle s Medium Invitrogen Cat 11995 Fetal bovine serum FBS Cat No TM999 500 Note does not need to be heat inactivated e 200 mM L Glutamine Sigma Cat No G7513 e Solution of 10 000 units ml Penicillin G sodium and 10 000 ug ml Strepto mycin sulphate Sigma Cat No P0781 Complete Medium DMEM supplemented with 100 units ml penicillin G sodium 100 ug ml streptomycin and 1076 fetal bovine serum FBS Puromycin Cat No CO21 e Polybrene Hexadimethrine Bromide Cat No G062 Trypsin EDTA Trypsin Sigma Cat No T3924 e Dulbecco s phosphate buffered saline DPBS VWR Cat No 82020 066 Tissue culture plates and flasks Storage e 293T Cells in Liquid Nitrogen Lentifectin at 4 C All other components at 20 C Spin briefly to recover contents and avoid repeated freeze thaw cycles Page 7 of 16 Lenti IlI HA Tag Handbook Protocol NOTE The following protocol is broken into sections for convenience However time should be taken to read through the full procedure before attempting Lenti Ill HA Construct Generation Generate a pLenti Ill HA expression vector con
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6. a full refund The information in this docu ment is subject to change without notice and should not be construed as a commitment by abm Inc orits affiliated corporations In no event shall abm Inc or its affiliated corporations be liable for incidental or consequen tial damages in connection with or arising from the use of this manual and product abm Inc products are warranted to meet our QC testing standards at the time of shipment Notice of problematic products must be made to abm Inc within 15 days of receipt of the product This product warranty limits abm Inc s liability to the replacement of the product only Technical Support For more information on abm Inc products please visit our website http www abmGood com For additional information or technical assistance please call or email us at Applied Biological Materials Inc Phone 604 247 2416 1 866 757 2414 Fax 604 247 2414 E mail technical abmGood com Page 1 of 16 Lenti IlI HA Tag Handbook Biosafety Our Lenti Ill HA Tag Expression System employs 3rd generation self in activating recombinant lentiviral vectors with enhanced biosafety and minimal relation to the wild type human HIV 1 virus The lentiviral particles produced with this system are replication incompetent and designed with a number of safety features to enhance its biosafety All Lentiviral Expression Systems provided from abm Inc include the following safety featu
7. GCT GGA TCC TCT AGA CTG Xhol HA Tag CAG CTC GAG TAC CCA TAC GAC GTC CCA GAC TAC GCT TGA 3 SV40 prom pLenti lll HA 8183bp rep origin Figure 2 Map of pLenti IlI HA Lenti Ill HA Tag Handbook Page 4 of 16 Bel vH III 3u91 Lenti Ill HA Tag Expression System The Lenti Ill HA Tag Expression System allows production of replication incompetent 3rd generation lentivirus that can stably transduce both dividing and non dividing mammalian cells with high efficiency Naldini 1998 and Dull et al 1998 Our lentiviral ex pression vector has been fully optimized for simple manipulations such as subcloning the gene of interest into our pLenti expression vector and easy viral DNA production including maxi DNA purification The vector simply works like any other plasmid In fact our vectors are so stable that non specific recombination or rearrangement in regular DH5a bacteria cells is rarely observed This is a significant advantage compared to similar lentiviral vectors offered by other companies which are associated with substantial adversity in subcloning and DNA production Because of our vector stability there is no need for special competent cells during transformation A general procedure for lentiviral production is shown in figure 1 on page 3 Our lentiviral vectors are 3rd generation and are compatible with packaging mixes that support the production of both 2nd and 3rd generation vectors Our optimized Lenti
8. Salon Lenti lll HA Tag Expression Systems Lenti IIl HA Tag Vector Complete Lenti Ill HA Tag Expression Kit Partial Lenti IlI HA Tag Expression Kit Supplemental Kit for All Lentivirus Systems Supplemental Kit for All Lentivirus Systems LV022 G327 G328 LV098 LV099 www abmGood com z E E w o 3 n o o amp a o 3 Table of Contents Notice to the Purchaser Technical Support Biosafety Protocol at a Glance Lenti Ill HA Tag Expression System General Information about Lentiviral Vectors How Lentiviruses Work VSV Glycoprotein Envelope Packaging Limits Materials Additional Materials Required Storage Protocol Packaging Mix 293T Cells Positive Control Transfection Procedure Concentrating Virus Long Term Storage Viral Titre Assays Transduction Procedure Troubleshooting References Contact Information Lenti IIl HA Tag Handbook O0 0 0 Oui NNN Ij C O o o o o oc a A o Notice to the Purchaser The products are for research use only Use in and or for diag nostics or therapeutics is strictly prohibited By opening and using the product the purchaser agrees to the following The components in this kit may not be distributed resold or modified for resale without prior written approval from Applied Biological Materials abm Inc If you do not agree to the above conditions please return the UNOPENED product to abm Inc within ten 10 days of receipt for
9. brene is a polycation that neutralizes charge interactions to increase binding between the pseu doviral capsid and the cellular membrane The optimal concentration of Polybrene depends on cell type and may need to be empirically de termined usually in the range of 1 8ug ml Excessive exposure to Poly brene gt 12 hr can be toxic to some cells Page 11 of 16 7 Lenti III HA Tag Handbook Protocol 3 5 Remove culture medium and replace with 0 5ml of complete DMEM medium with 10 serum and Polybrene from Step 2 For each viral stock use three wells Infect MDA MB 468 cells by adding lul of viral stock into the first well dilution factor of 500 10ul of viral stock into the second well dilution factor of 50 and 100ul of viral stock into the last well dilution factor of 5 For controls add 0 5ml of DMEM medi um with Polybrene from Step 2 Incubate at 37 C with 5 CO over night Remove culture medium and replace with 1ml of complete DMEM medium without Polybrene Incubate the cells at 37 C with 576 CO overnight The following day split the cells 1 3 to 1 5 if necessary depend ing on the growth rate of cells Incubate in complete D MEM for an ad ditional 24 48 hours Count the fraction of fluorescent cells by FACS analysis You may also count the GFP positive cells under a fluorescent microscope but the results may be less accurate due to inconsistencies in counting methods Use an average of the fract
10. ion of GFP cells in 5 10 random fields to estimate the overall percentage of GFP cells on the plate Multiply the number of infected cells by 1 5x10 in this example the expected num ber of MDA MB 468 cells on the plate at the moment of infection and by The corresponding dilution factor then divide by 0 5ml to determine the relative titre of the virus in the supernatant Alternatively the viral titre can also be estimated by real time PCR using ABM s Lentiviral qPCR Titre Kit Cat No LV500 or p24 based ELISA titre kit Cat No LV501 E Transduction Procedure The following information should be considered before one attempts target cell transduction The transduction efficiency of target cells varies significantly under different experimental conditions including virus concentration exposure time to virus and growth area of cells To determine the viral concentration required to provide the desired multiplicity of infection MOI for your target cells perform several transductions with different concentrations of viral par ticles containing GFP control plasmid Results from these test transductions should be used to determine an optimal concentration that yields the high est percentage of infected cells Lenti Ill HA Tag Handbook Page 12 of 16 S ke lt T m c o gt i D 5 ml F T T gt a Q Protocol Recombinant gene expression can be measured directly 48 72 hours after transducti
11. ion step to remove any remaining cellular debris The viral supernatant can be concentrated using the protocols discussed in the following section if higher fitre virus is required B Concentrating Virus There are many protocols that have been established to concentrate VSVG pseudotyped lentiviruses without significantly affecting their ability to transduce target cells These include ultracentrifugation Yee 1999 filter based ion exchange chromatography Ultra Pure Cat No LV998 and size exclusion chromatography Speedy Lentivirus Purification Cat No LV999 However we would strongly recommend using abm s Ultra Pure Lentivirus Pu rification Kit Cat No LV998 for pLenti miR vector concentration and purifica tion based on our in house testing data Lenti Ill HA Tag Handbook Page 10 of 16 2 ct ae T am gt a tel Protocol C Long Term Storage Viral stocks stored at 80 C should be stable for at least one year Re peated freeze thaw cycles will result in a loss of viral titre Based on our in house data each freeze thaw will lead to a 2576 loss of viral titre D Viral Titre Assays It is useful to titre the viral supernatant before proceeding with the trans duction experiments for the following reasons To ensure that viral stock is viable To determine the percentage of target cells that can be transduced with the pseudoviral stock To control the number of copies of viral construc
12. ition include a transduction well with GFP positive control virus and other appropriate positive and negative control viral constructs Incubate cells at 37 C with 576 CO overnight Remove the culture medium and replace with 1ml of complete medium Incubate cells at 37 C with 5 CO overnight The following day split the cells 1 3 to 1 5 depending on the growth rate of your target cells and continue incubating for 48 hours in complete DMEM The infected target cells can be either analyzed for transient ex pression or selected for stable expression using appropriate selection markers Page 13 of 16 Lenti Ill HA Tag Handbook Troubleshooting Problem Possible Cause Solution No Viral Particles Lenti IlI HA Tag DNA modified e g acetylation or methylation Re transform plasmid into an authetic DH5a Low Viral Titre Low transfection efficiency poor quality DNA low 293T viability transfection media contain ing antibiotics and serum Low transfection efficiency Insufficient DNA used for transfection 293T cell density too low Viral supernatan har vested too early Viral supernatant subjected to multiple freeze thaw cycles Polybrene not used dur ing transduction Purify DNA with an endotox in free Maxi colomn Use 293T cells under passage 16 Reduce transfection antibi otic or serum efficiency Optimize calcium transfection procedure Use 30 50ug of ex
13. k Protocol g Add 0 65ml serum to each transfected culture dish and return the dishes to incubator Incubate overnight 3 Thefollowing day Day 3 remove the medium containing the Lenti fectin DNA complexes and replace with 10ml complete culture medium Incubate at 37 C in a humidified 576 CO incubator Note Expression of the VSVG glycoprotein can cause 293T cells to fuse resulting in the ap pearance of large multinucleated cells known as syncytia This morpho logical change is normal and does not affect production of the lentivi rus S ke lt x af mm E C E 4 Harvest virus containing supernatants 48 72hrs post transfection Day 4 5 by collecting medium into to a 15ml sterile capped conical tube Caution Remember that you are now working with infec tious virus at this stage Follow the recommended guidelines for working with BL 2 organisms see page 3 for more informa tion 5 Centrifuge supernatants at 3000rpm for 15 min at 4 C to pellet de bris Optional Filter the viral supernatant through 0 45um PVDF syringe filter Millipore Cat No SLHVR25LS 6 Aliquot viral supernatants into cryovials in 1 0ml portions and Store viral stocks at 80 C Proceed to titre your lentiviral stock page 10 Nofe If you plan to use your lentiviral construct for in vivo applica tions we recommend filtering your viral supernatant through a sterile 0 45um low protein binding filter after the low speed centrifugat
14. ls Curr Opin Biotechnol 9 457 463 Naldini L 1999 in The Development of Human Gene Therapy Friedmann T ed pp 47 60 Cold Spring Harbor Laboratory Press Cold Spring Harbor NY Sambrook J amp Russell D W 2001 Molecular Cloning A Laboratory Manual Cold Spring Harbor Laboratory Press Cold Spring Harbor NY 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 Ret roviral 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 Fried mann T 1987 Gene Expression from Transcriptionally Disabled Retroviral Vectors Proc Natl Acad Sci USA 84 5197 5201 Page 15 of 16 Lenti IlI HA Tag Handbook Contact Information Applied Biological Materials Inc Website www dbmGood com Phone 8 30am 4 30pm PST M F Toll Free 1 866 757 2414 Local 604 247 2416 Fax 604 247 2414 24Hr Address Email General Information info abmGood com Order Products order abmGood com Technical Support technical abmGood com siRNA siRNA abmGood com Suite 8 13520 Crestwood Place Richmond BC Canada Distributors
15. lybrene dur ing transduction Use minimum antibiotics for ef fective selection Try a different cell line Lenti IlI HA Tag Handbook Page 14 of 16 References 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 Pseudotyped Retroviral Vectors Concentration to a Very High Titer and Efficient Gene Transfer into Mammalian and Non mammalian Cells Proc Natl Acad Sci USA 90 8033 8037 Dull T Zufferey R Kelly M Mandel R J Nguyen M Trono D and Na Idini L 1998 A Third Generation Lentivirus Vector with a Conditional Pack aging 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 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 J L Monath T P Roizman B and Straus S E eds 3rd Ed pp 1881 1975 Lippincott Raven Publishers Philadelphia PA Naldini L 1998 Lentiviruses as Gene Transfer Agents for Delivery to Non dividing Cel
16. on transient transduction but selecting stably trans duced cells will require additional time after transduction The decision to use transiently transduced cells or selected stable cells will depend on the nature of your target cells biological assay and transduction efficiency For efficient transducable cells e g 293 HT1080 HeLa MDA MB 468 cells etc most biological assays can be performed following transient transduc tion However for difficult to transduce cells it is desirable to select the clones that stably expresses the Lentivector construct for experimental as says The following provides general guidelines as a starting point for deter mining optimal conditions for target cell transduction 1 Plate target cells in a 24 well plate 24 hours prior to viral infection at a density of 0 5x105 cells per well Add 0 5 ml of complete optimal medium with serum and antibiotics and incubate cells at 37 C with 5 CO overnight Note It is possible to use other plate formats for trans duction In this case the amount of cells should be adjusted depend ing on the growth area of the well plate Prepare a mixture of complete media with Polybrene at a concentration of 2ug ml Remove media from the wells and re place with 0 5ml of the Polybrene media mixture per well for 24 well plate Infect target cells by adding several different amounts of viral stock example lul 5ul 1Oul and 100ul of virus In add
17. pression vector and 30 50ug of packaging mix Optimal cell density is 90 9576 Optimal viral titres can be collected 48 72 hours post transfection Each freeze thaw cycle can lose 2576 of the titre Make ali quots for long term storage Transduce cells in the pres ence of polybrene No Transgene Expression Promoter silencing MOI too low Viral stocks stored incorrectly Target cells not transduc ible with lentiviral vectors Antibiotic concentration too high Cells harvested too early for assay Lentiviral vector may integrate into a chromosomal region that silences the promoter Screen multiple antibiotic resistant clones and select the one with the highest expresssion levels Use maximum MOI for cell transduction This is very critical for Tet expression vectors Aliquot and store at 80C Avoid freeze thaw cycles Transduce target cells in the presence of polybrene Determine antibiotic sensitivity of target cells by performing o killing curve Use mini mum antibiotic concentration required Perform expression assay 3 4 days post transduction and induction to allow the accumulation of expressed protein Cytoxic Effects of Target Cells Large volume of viral superna tant used for transduction Polybrene concentration too high Antibiotic concentration too high Gene of interest toxic to cells Dilute viral supernatant 1 2 to 1 3 during transduction Use less or omit po
18. res An enhancer deletion in the U3 region of 3 ALTR ensures self inactivation of the lentiviral vector following transduction amp integration into the target cell s genomic DNA Utilization of a RSV promoter upstream of S ALTR allows efficient Tet inde pendent production of viral RNA The number of lentiviral genes necessary for packaging replication and transduction is limited to three Gag Pol Rev and their expression is de rived from different plasmids all lacking packaging signals The plasmids share no significant homology to the expression vector preventing the gen eration of replication competent virus None of the Gag Pol or Rev genes will be present in the packaged viral genome thus making the mature virus replication incompetent Despite the safety features discussed above it is highly recommend ed that all manipulation with lentiviral vectors including viral production and transduction be performed under Biosafety Level 2 BL 2 All published BL 2 guidelines with proper waste decontamination should be strictly fol lowed In addition exercise extra caution when creating lentivirus carrying potentially harmful or toxic genes e g oncogenes For more information about the BL 2 guidelines and lentivirus handling refer to Biosafety in Mi crobiological and Biomedical Laboratories 5th Edition This may be down loaded at www cdc gov biosafety publications bmbl5 index htm It is also important to consul
19. sfection Day 1 plate 293T cells in a 10cm tissue culture plate so that they will be 90 95 confluent on the day of transfection i e 5x106 cells in 10ml of growth medium containing se rum As arule one 15cm culture dish at 95 confluence can be subcul tured into five 10cm dishes while one 10cm dish at 95 confluence can be subcultured to two 10cm dishes On the day of transfection Day 2 set up the transfection mix a Ina sterile 15ml culture tube dilute 15g of Lenti Combo Mix and 10ug of pLenti expression plasmid DNA in 1 0ml of medium without serum Mix gently b In a separate sterile 15ml tube dilute 80pl of Lentifectin mix gently before use in 1 0ml of medium without serum Mix gently and in cubate for 5 minutes at room temperature c After the 5 minutes of incubation combine the diluted DNA with the diluted Lentifectin Mix gently d Incubate for 20 minutes at room temperature to allow the Lentifectin DNA complexes to form e Add 4 5ml serum free medium to the complexes followed by gently mixing f Remove the medium from the cells and then add Lentifectin DNA complexes carefully to culture dishes without dislodging cells In cubate the cells for 5 8 hours at 37 C in a humidified 576 CO incubator Note 293T cells are poorly adhesive to most culture dishes It is always recommended to add or change medium against the wall of culture dishes to avoid dislodging cells Page of 16 Lenti IlI HA Tag Handboo
20. t with the health and safety officer s at your institution for guidelines regarding the use of lentiviruses and to always follow standard microbiological practices which include Wear gloves and a lab coat at all times e Always work with pseudoviral particles in a Class Il culture facility and that all procedures are performed carefully to minimize splashes and aerosols Work surfaces are decontaminated at least once a day and after any spills of viable material All cultures stocks and other regulated wastes are decontaminated be fore disposal by an approved decontamination method like autoclaving Lenti Ill HA Tag Handbook Page 2 of 16 Protocol at a Glance Packaging Mix Expression Vector 293T Cells Step 1 Co transfect 293T cells with a lentiviral vector and pack aging mix Pseudoviral Particles Step 2 m m Collect viral particles and ee determine titre Target Cells Step 3 Infect Target Cells Target Cells Transduced Step 4 Assay Cells Figure 1 Procedure for Transient Production of Pseudoviral Particles and Transduction of Target Cells Page 3of 16 Lenti IIl HA Tag Handbook Nhel Xmal Clal Mfel Scal 5 GCT AGC CCC GGG ATC GAT CAA TTG AGT ACT SnaBl Kpnl TAC GTA GGT ACC cea x TGG TGG CCT GCA GGT EcoRI Spel Stul Sall EcoRV GAA TE ACT AGT ACC GGT AGG CCT GTC GAC GAT Apal Not BamHI Xbal ATC GGG CCC GCG GCC
21. taining your gene of in terest For information on subcloning using the multiple cloning sites available in our lentiviral vectors refer to a standard molecular cloning manual Sambrook J amp Russell D W Once the expression construct has been produced perform a Maxi DNA purification for transfection Packaging Mix All plasmids required for the production of recombinant lentiviruses are provided in optimized mixtures We have developed 2 different packaging mixes for producing recombinant lentiviral particles in either 2nd or 3rd genera tion lentiviral vectors 2nd Generation Packaging System Mix Cat No LV003 is used for the production of 2nd generation lentiviral particles and 3rd Generation Packaging System Mix Cat No LV053 is used for the packaging of 3rd genera tion lentiviral particles All lentiviral expression vectors provided by abm Inc are of 3rd generation and can be packaged by either 2nd or 3rd Generation Pack aging Mixes In general relatively higher titres can be achieved with the 2nd Generation Packaging System Mix but lentiviral particles packaged with the 3rd Generation Mix have a higher safety profile 293T Cells The 293T cell line is widely used for optimal cell line for lentivirus produc tion Naldini et al 1996 The health of 293T cells at the time of transfection is a critical factor for the success of lentivirus production The use of unhealthy cells will negatively affect the transfec
22. tion efficiency resulting in lower titre lentiviral stocks For optimal lentivirus production follow the guidelines below to culture 293T cells before use in transfection ensure cell viability is greater than 90 e subculture and maintain cells in complete medium containing 0 1mM MEM Non Essential Amino Acids 4mM L Glutamine ImM sodium pyruvate 1076 FBS do not allow cells to overgrow before passaging use cells that have been subcultured for less than 16 passages Positive Control o We recommend using a positive control vector such as pLenti GFP to generate a control lentiviral stock that can be used to help you optimize expres sion conditions in your target cells Lenti Ill HA Tag Handbook Page 8 of 16 T T lt L T T ET Pur D E ct T5 I gt a Ke Protocol A Transfection Procedure We produce lentiviral stocks in 293 cells using the following opti mized transfection conditions The amount of lentivirus produced using these recommended conditions 10ml of virus at a titre of at least 1x105trans ducing units TU ml is generally sufficient to transduce at least 1x106 cells at a multiplicity of infection MOI of 1 higher titre lentivirus can be produced by scaling up transfection For example 10 wells of cells plated at 1x105 cells well in 6 well plates could each be transduced with 1ml of a 1x10 TU ml virus stock to achieve an MOI of 1 1 One day before tran
23. ts per target cell The commonly used protocol for measuring relative titres uses a positive control expression plasmid i e GFP mixed with expression construct as an internal control at a ratio of 1 100 and is packaged into pseudo viral particles In an alternative approach the GFP control plasmid can be packaged separately but in parallel with your construct as an external con trol In this scenario the control plasmid can be used to check and optimize the transfection packaging steps see transfection procedure Recently other in vitro protocols including qRCR and HIV p24 protein based ELISA have been developed for quick assays To determine the relative titre transduce a target cell line like MDA MB 468 in the presence of Polybrene 2ug ml for 12 16 hrs and then count the number of cells expressing GFP either by fluorescence microscopy or FACS 1 Foreach viral stock plate MDA MB 468 cells one day prior to viral infection in a 24 well plate at a density of 0 6 1x10 cells per well Add 1ml of complete D MEM medium with serum and antibiotics and incubate cells at 37 C with 576 CO overnight Note It is possible to use bigger cul ture dishes for transduction especially when a large number of cells is needed for FACS analysis In this case the amount of cells should be ad justed based on the growth area of the dish 2 Prepare complete D MEM medium plus 1076 FBS with Polybrene to a final concentration of 2ug ml Note Poly
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