Vivid Colors pLenti6.2- GW/EmGFP
Contents
1. 80 C See the next page for further details about long term virus stock storage An alternative transfection procedure is provided below to cotransfect 293FT cells Note that use of this procedure generally results in production of lentiviral stocks with a slightly lower titer than those produced when using the 293FT Transfection Protocol previous page 1 The day before transfection plate the 293FT cells in a 10 cm tissue culture plate such that they will be 90 95 confluent on the day of transfection i e 6 x 10 cells in 10 ml of growth medium containing serum 2 Onthe day of transfection remove the culture medium from the 293FT cells and replace with 5 ml of growth medium or Opti MEM I Medium containing serum Do not include antibiotics in the medium 3 Prepare DNA Lipofectamine 2000 complexes as instructed in the 293FT Transfection Protocol Step 1 previous page 4 Add the DNA Lipofectamine 2000 complexes dropwise to each plate of cells Mix gently by rocking the plate back and forth Incubate the cells overnight at 37 C in a CO incubator Follow Steps 8 11 as instructed in the 293FT Transfection Protocol above If you plan to use your EmGFP lentivirus for in vivo applications we recommend filtering your viral supernatant through a sterile 0 45 um low protein binding filter after the low speed centrifugation step see Step 10 above to remove any remaining cellular debris We recommend using Millex HV 02. biohazard concerns Depending on how you wish to determine transfection efficiency you should follow the recommendations in the transfection protocol on page 9 Materials Needed You will need the following items e pLenti6 2 GW EmGIP Expression Control Vector 0 5 ug ul Materials available separately see page vi e ViraPower Packaging Mix 1 ug ul e 293FT cells 6 x 10 cells for each transfection e Complete growth medium for 293FT cells D MEM containing 10 FBS 2mM L glutamine 0 1 mM MEM Non Essential Amino Acids 1 penicillin streptomycin and 1 mM MEM Sodium Pyruvate Note MEM Sodium Pyruvate provides an extra energy source for the cells and is available from Invitrogen page vi e Lipofectamine 2000 transfection reagent mix gently before use e Optional Irrevelant plasmid such as an empty DEST vector 1 ug ul if you do not intend to make virus producing cells e Opti MEM I Reduced Serum Medium pre warmed to 37 C e Fetal Bovine Serum e Sterile 10 cm tissue culture plates e Sterile tissue culture supplies e 15mlsterile capped conical tubes e Cryovials e Optional Millex HV 0 45 um PVDF filters Millipore cat no SLHVR25LS or equivalent to filter viral supernatants e Inverted fluorescence microscope with FITC filter or Omega XF100 filter see next page for detecting EmGFP expressing cells in culture or a flow cytometry system with a FITC filter to quantitatively detect EmGFP expre
3. you are ready to optimize the transduction conditions for mammalian cell line of choice Reminder Remember that the pLenti6 2 GW EmGFP Expression Control Vector contains a deletion in the 3 LTR that leads to self inactivation of the lentivirus after transduction into mammalian cells Once integrated into the genome the lentivirus can no longer produce packageable virus After transducing your mammalian cell line of choice with the pLenti6 2 GW EmGFP Expression Control Vector you can assay for expression of EmGFP by either transient expression or stably transduced cells by performing one of the following e Poola heterogeneous population of cells and test for EmGFP expression 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 optimal detection of EmGFP e Select for stably transduced cells using Blasticidin This requires a minimum of 10 12 days after transduction but allows generation of clonal cell lines that stably express EmGFP Note We have observed stable expression of EmGFP for at least 6 weeks following transduction and selection To obtain optimal expression of your gene of interest you will need to transduce the EmGFP lentivirus into your mammalian cell line of choice using a suitable MOI MOT is defined as the number of virus particles per cell and generally correlates with the number of integration ev
4. 2 GW EmGFP Expression Control Vector in 10 mM Tris HCl 1 mM EDTA pH 8 0 Accessory Products Additional Products vi Additional products available from Invitrogen are listed below For more information visit our website at www invitrogen com or contact Technical Support page 27 Product Amount Catalog no ViraPower Lentiviral Gateway Expression Kit 1 kit K4960 00 ViraPower UbC Lentiviral Gateway 1 kit K4990 00 Expression Kit ViraPower II Lentiviral Gateway Expression 1 kit K367 20 Kit ViraPower II Lentiviral C Lumio Gateway 1 kit K370 20 Expression Kit ViraPower II Lentiviral N Lumio Gateway 1 kit K371 20 Expression Kit pLenti6 V5 Directional TOPO Cloning Kit 1 kit K4950 00 pLenti6 3 V5 GW EmGFP Expression Control 20 pg 40 pl of 0 5 ng ul V370 06 Vector in TE Buffer pH 8 0 ViraPower Bsd Lentiviral Support Kit 20 reactions K4970 00 ViraPower Lentiviral Packaging Mix 60 reactions K4975 00 293FT Cell Line 3 x 10 cells R700 07 Opti MEM I Reduced Serum Medium 100 ml 31985 062 500 ml 31985 070 One Shot StbI3 Chemically Competent E coli 20 x 50 ul C7373 03 S N A P Midiprep DNA Isolation Kit 20 reactions K1910 01 PureLink HiPure Plasmid Midiprep Kit 25 reactions K2100 04 50 reactions K2100 05 Lipofectamine 2000 Transfection Reagent 0 75 ml 11668 027 1 5 ml 11668 019 Blasticidin 50 mg R210 01 Ampicillin 5g Q100 16 Overview Description Fe
5. 603 7200 F 1 760 602 6500 E tech_support invitrogen com For country specific contact information visit our web site at www invitrogen com
6. Cloning Products Gateway Clone Distribution Policy Limited Use Label License No 51 Blasticidin amp the Blasticidin Selection Marker 28 Use of the pLenti6 2 GW EmGFP Expression Control Vector is covered under the licenses detailed below The purchase of this product conveys to the buyer the non transferable right to use the purchased amount of the product and components of the product in research conducted by the buyer whether the buyer is an academic or for profit entity The 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 ClonaseTM 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 materials made by the employment of this product or its components for Commercial Purposes The buyer may transfer information or m
7. base 520 HIV 1 psi y packaging signal bases 521 565 HIV 1 Rev response element RRE bases 1075 1308 3 splice acceptor base 1656 3 splice acceptor base 1684 CMV promoter bases 1809 2392 attB1 site bases 2440 2464 EmGFP bases 2470 3189 attB2 site bases 3190 3214 PGK promoter bases 3336 3841 EM7 promoter bases 3852 3918 Blasticidin resistance gene bases 3919 4317 AU3 HIV 1 3 LTR bases 4403 4637 AU3 bases 4403 4456 Truncated HIV 1 3 LTR bases 4457 4637 SV40 polyadenylation signal bases 4709 4840 bla promoter bases 5699 5797 Ampicillin b a resistance gene bases 5798 6658 pUC origin bases 6803 7476 Continued on next page 25 Features of pLenti6 2 GW EmGFP Expression Control Vector Features of The pLenti6 2 GW EmGFP Expression Control Vector contains the following pLenti6 2 elements All features have been functionally tested and the vector has been fully GW EmGFP sequenced Feature Benefit Rous Sarcoma Virus RSV Allows Tat independent production of viral mRNA enhancer promoter Dull et al 1998 HIV 1 truncated 5 LTR Permits viral packaging and reverse transcription of the viral mRNA Luciw 1996 5 splice donor and 3 acceptors Enhances the biosafety of the vector by facilitating removal of the 8 packaging sequence and RRE such that expression of the gene of interest in the transduced host cell is no longer Rev dependent Dull et al 1998 HIV 1 psi y8 packag
8. 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 containing clones and facilitate their use of this powerful technology in their research Use of Invitrogen s Gateway Technology includi
9. pose some biohazardous risk since it can transduce primary human cells For this reason we highly recommend that you treat lentiviral stocks as Biosafety Level 2 BL 2 organisms and strictly follow all published BL 2 guidelines with proper waste decontamination For more information about BL 2 guidelines and lentivirus handling refer to the document Biosafety in Microbiological and Biomedical Laboratories 4 Edition published by the Centers for Disease Control CDC This document may be downloaded at the following address http www cdc gov od ohs biosfty bmbl4 bmbl4toc htm Handle all lentiviruses in compliance with established institutional guidelines Since safety requirements may vary at individual institutions we recommend consulting the health and safety guidelines and or officers at your institution prior to use of the ViraPower Lentiviral Expression System More information about the specific biosafety features of the ViraPower Lentiviral Expression System can be found in the System manual The pLenti6 2 GW EmGFP Expression Control Vector is supplied in solution as 40 ul of 5 ug ul control vector in 10 mM Tris HCl 1 mM EDTA pH 8 0 You can use this solution for production of lentivirus or you can propagate and maintain the plasmid as described below If you wish to propagate and maintain the pLenti6 2 GW EmGFP Expression Control Vector we recommend using 10 ng of the vector to transform One Shot StbI3 C
10. 0 603 7200 Fax 760 602 6500 continued on next page Purchaser Notification Continued Limited Use Label License No 108 Lentiviral Technology Limited Use Label License No 109 Retroviral Helper Lines Limited Use Label License No 127 GFP with Heterologous Promoter 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 fluores cent proteins The Lentiviral Technology based upon the lentikat system is licensed from Cell Genesys Inc under U S Patent Nos 5 834 256 5 858 740 5 994 136 6 013 516 6 051 427 6 165 782 and 6 218 187 and corresponding patents and applications in other countries for internal research purposes only Use of this technology for gene therapy applications or bioprocessing other than for non human research use requires a license from Cell Genesys Cell Genesys Inc 342 Lakeside Drive Foster City California 94404 The purchase of this product conveys to the buyer the non transferable right to use the purchased amount of the product and components of the product in research conducted by the buyer whether the buyer is an academic or for profit entity including non gene therapy research and target validation applications in laboratory animals Retroviral helper cell lines are licensed from Wisconsin Alumni Research Foundation under U S Patents and corresponding patents and a
11. 45 um PVDF filters Millipore Catalog no SLHVR25LS for filtration If you wish to concentrate your viral stock to obtain a higher titer perform the filtration step first before concentrating your viral stock see page 19 Continued on next page Producing EmGFP Lentivirus in 293FT Cells Continued Long Term Aliquot lentiviral stocks in cryovials at 80 C for long term storage Repeated Storage freezing and thawing is not recommended as it may result in loss of viral titer When stored properly viral stocks of an appropriate titer should be suitable for use for up to one year After long term storage we recommend re titering your viral stocks before transducing your mammalian cell line of interest Scaling Up Virus It is possible to scale up the cotransfection experiment to produce a larger Production volume of lentivirus if desired For example we have scaled up the cotransfection experiment from a 10 cm plate to a T 175 cm flask and harvested up to 30 ml of viral supernatant If you wish to scale up your cotransfection remember that you will need to increase the number of cells plated and the amounts of DNA Lipofectamine 2000 and medium used in proportion to the difference in surface area of the culture vessel 11 Titering EmGFP Lentivirus Introduction Experimental Outline Note Range of Dilutions Selecting a Cell Line 12 After you have produced your EmGFP lentiviral stock in 293FT cells
12. Cell Line manual Do not allow cells to overgrow before passaging e Use cells that have been subcultured for less than 20 passages The fluorescent signal from EmGFP can be detected with standard FITC filter sets However for optimal detection of the fluorescent signal you may use the Omega XF100 filter set for your cell culture inverted microscope that is optimized for detection of EmGFP Fluorescent Excitation Emission Filter Set for Fluorescence Protein nm Microscopy EmGFP 487 509 Omega XF100 For information on obtaining this filter set contact Omega Optical Inc www omegafilters com Continued on next page Producing EmGFP Lentivirus in 293FT Cells Continued ViraPower Packaging Mix Lipofectamine 2000 Opti MEM TM The ViraPower Packaging Mix contains a mixture of plasmids pLP1 pLP2 and pLP VSVG which are cotransfected into 293FT cells to supply the structural and replication proteins in trans for producing lentivirus TM The ViraPower packaging Mix is supplied with the ViraPower Lentivirus Support Kits or is available separately from Invitrogen page vi To prepare the stock solution of the ViraPower Packaging Mix resuspend the contents of the tube 195 ug in 195 ul of sterile water to obtain a 1 ug ul stock The Lipofectamine 2000 reagent Ciccarone et al 1999 is a proprietary cationic lipid based formulation for optimal transfection of nu
13. Invitrogen Vivid Colors pLenti6 2 GW EmGFP Lentiviral expression plasmid containing EmGFP for optimization of lentivirus production titer and transduction using the ViraPower Lentiviral Expression System Catalog no V369 20 Version C 14 December 2010 25 0861 A Limited Label License covers this product see Purchaser Notification By use of this product you accept the terms and conditions of the Limited Label License Table of Contents Kit Contents and Re v Accessory Products aaan sedan Leere REOR E ete vi Introduction sees d 1 Let neten beten beende dee ee ents 1 Methods Ee 5 Producing EmGFP Lentivirus in 293FT Cells EEN 5 Titering EmGEP Lenfivirus E 12 Transduction and Analysis so tenen oni eo beide eh anal ash base 18 Troubleshooting stenen cuve ran en BID UBER deele 21 ADPONGIX quc EP 23 CD CA 23 Map of pLenti6 2 GW EmGFP Expression Control Vector unuunanenenenenenenerseneneneneneeenenenenenennenenen 25 Technical Support eite medo md mean esce 27 Purchaser Notification sto ee ee e eei ete pt etes ha I t ee HL IHR dre a i ITE 28 Gateway Clone Distribution Policy u 31 References cioe ee Om onte ipte ue PIRE HD tete ERO ete tote tefie i pter an Ee 32 iii iv Kit Contents and Storage Shipping and The pLenti6 2 GW EmGFP Expression Control Vector is shipped on wet ice Storage Upon receipt store at 20 C Contents 20 ug of control vector is supplied in solution as 40 ul of 0 5 pg pl pLenti6
14. U ml To obtain higher lentivirus titer you can concentrate your virus see page 19 The titer of concentrated lentivirus stocks may be up to 1 x 10 TU ml Continued on next page Titering EmGFP Lentivirus Continued Alternate Protocol Itis possible to estimate EmGFP lentiviral titer by counting EmGFP positive cells for Titering using fluorescence microscopy see page 7 for details Note that this method is EmGFP Lentivirus labor intensive and the results are much less accurate than using the flow cytometry method because of the necessity for a smaller sample size and reliance on visual discrimination of EmGFP positive cells 1 Using fluorescence microscopy determine two dilutions that have a countable number of EmGFP positive cells i e 100 or fewer 2 Countthe total number of cells for each dilution the well may be divided into quarters to facilitate counting and determine the number of EmGFP positive cells 3 Calculate the titer by taking the average of the titers from the 2 wells For example if the 10 dilution has 46 green cells in the well and the 10 dilution has 5 green cells in the well the titer would be 4 8 x 10 TU ml average of 46 x 10 and 5 x 10 17 Transduction and Analysis Introduction Evaluating Transduction with EmGFP Expression Multiplicity of Infection MOI Determining the Optimal MOI 18 Once you have generated an EmGFP lentiviral stock with a suitable titer
15. aterials 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 infringement of the above patents based upon the manufacture use or sale of a therapeutic clinical diagnostic vaccine or prophylactic produc
16. atures of the Vector Introduction The Vivid Colors pLenti6 2 GW EmGFP Expression Control Vector contains the Emerald Green Fluorescent Protein EmGFP under the control of a constitutive promoter and viral elements that allow packaging of the control plasmid into virions pLenti6 2 GW EmGFP is designed for use with the ViraPower or ViraPower II Lentiviral Expression System for the following applications e Asa positive transfection control for 293FT cells e As a titer control to produce an EmGFP expressing lentivirus stock e Asa transduction control to help you determine optimal lentiviral transduction conditions for your target mammalian cell line The pLenti6 2 GW EmGFP Expression Control Vector contains the following elements e Rous Sarcoma Virus RSV enhancer promoter for Tat independent production of viral mRNA in the producer cell line Dull et al 1998 e Modified HIV 1 and 3 Long Terminal Repeats LTR for viral packaging and reverse transcription of the viral mRNA Dull et al 1998 Luciw 1996 Note The U3 region of the 3 LTR is deleted AU3 and facilitates self inactivation of the 5 LTR after transduction to enhance the biosafety of the vector Dull et al 1998 e HIV 1 psi Y packaging sequence for viral packaging Luciw 1996 e HIV Rev response element RRE for Rev dependent nuclear export of unspliced viral mRNA Kjems et al 1991 Malim et al 1989 e Human cytomegalovirus immediate ear
17. chim Biophys ACTA 1219 653 659 Kjems J Brown M Chang D D and Sharp P A 1991 Structural Analysis of the Interaction Between the Human Immunodeficiency Virus Rev Protein and the Rev Response Element Proc Natl Acad Sci USA 88 683 687 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 Malim M H Hauber J Le S Y Maizel J V and Cullen B R 1989 The HIV 1 Rev Trans activator Acts Through a Structured Target Sequence to Activate Nuclear Export of Unspliced Viral mRNA Nature 338 254 257 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 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 Shimomura O Johnson F H and Saiga Y 1962 Extraction Purification and Pr
18. cleic acids into eukaryotic cells The recommended procedure to co transfect 293FT cells differs from the traditional Lipofectamine 2000 transfection procedure in that you will 1 First prepare DNA Lipofectamine 2000 complexes and add them to plates containing growth media then 2 Add the 293FT cells to the media containing DNA Lipofectamine 2000 complexes and allow the cells to attach and transfect overnight see detailed procedure on the next page Using this procedure we consistently obtain lentiviral stocks with titers that are 3 to 4 fold higher than lentiviral stocks generated using the traditional Lipofectamine 2000 transfection procedure TM TM Lipofectamine 2000 is supplied with the ViraPower Lentiviral Support Kits or is available separately from Invitrogen page vi TM To facilitate the optimal formation of DNA Lipofectamine 2000 complexes we recommend using Opti MEM I Reduced Serum Medium available from Invitrogen page vi Continued on next page Producing EmGFP Lentivirus in 293FT Cells Continued 293FT Transfection Protocol Follow the protocol below to transfect 293FT cells with the pLenti6 2 GW EmGFP Expression Control Vector Note that this protocol differs from the transfection protocol in the ViraPower System manual so use the protocol below only for producing EmGFP lentivirus 1 Prepare DNA Lipofectamine 2000 complexes In a sterile 15 ml tube combine one
19. ct 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 Purpose and that such collaborator agrees in writing a not to transfer such materials to any third party and b to use such transferred materials and or information solely for research and not for Commercial Purposes Commercial Purposes means any activity by a party for consideration and may include but is not limited to 1 use of the product or its components in manufacturing 2 use of the product or its components to provide a service information or data 3 use of the product or its components for therapeutic diagnostic or prophylactic purposes or 4 resale of the product or its components whether or not such product or its components are resold for use in research 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 o
20. cytometry you can use 2 Note formaldehyde or paraformaldehyde in calcium magnesium free PBS However these fixatives may increase autofluorescence of the cells thus it is critical to include fixed mock transduced cells as a negative control for flow cytometry detection parameters Preparing Cells Prepare cells for flow cytometry using a FITC filter according to the established for Flow protocols in use at your flow cytometry facility Refer to page 4 for specific Cytometry excitation emission properties of EmGFP The steps below provide simple guidelines and other methods may be suitable 1 At day 4 post transduction dissociate the cells from the plate by using trypsin or cell dissociation buffer 2 Spin the cells at low speed to remove residual media components and resuspend the cell pellet in flow cytometry buffer such as calcium magnesium free PBS with 1 FBS at the required density for analysis on your flow cytometer Fixing the cells is not necessary but may be done see Note above 3 Use the mock transduced cells and the lowest dilution of virus i e 107 as the negative and positive samples respectively to set up the parameters of your flow cytometer Continued on next page 15 Titering EmGFP Lentivirus Continued Calculating Lentiviral Titer What You Can Expect 16 EmGEFP lentivirus titers should be calculated from the dilutions at which the percentage of GFP positive cells fall within the range
21. e page 13 to a final volume of 1 ml Important Do NOT dilute virus in culture medium containing Blasticidin Note You may prepare a wider range of serial dilutions e g 107 to 10 over several 6 well plates of cells or perform multiple replicates if desired Remove the culture medium from the cells Mix each virus dilution gently by inversion DO NOT vortex and add to each well of cells Swirl the plate gently to mix Incubate at 37 C in a CO incubator overnight The following day Day 3 remove the virus containing media from the plates and discard See Caution previous page Replace with 2 ml of complete culture medium and return cells to the 37 C CO incubator overnight Important Do NOT add Blasticidin to the culture medium At 4 days post transduction Day 6 determine the EmGFP lentivirus titer You may confirm EmGFP expression by visualizing the cells using a fluorescence microscope See page 7 for recommended filter sets If determining titer by flow cytometry see Preparing Cells for Flow Cytometry next page Note If you do not have access to a flow cytometry facility you may estimate EmGFP lentiviral titer by an alternate method using fluorescence microscopy on page 17 You may also determine titers using the Blasticidin selection protocol which is described in the ViraPower System Manual Continued on next page Titering EmGFP Lentivirus Continued If you wish to fix your cells before flow
22. ecommend using the same mammalian cell line to titer your lentiviral stock as you will use to perform your expression studies However in some instances you may wish to use a different cell line to titer your lentivirus e g if you are performing expression studies in a non dividing cell line or a primary cell line In these cases we recommend that you choose a cell line with the following characteristics to titer your lentivirus e Grows as an adherent cell line e Easy to handle e Exhibits a doubling time in the range of 18 25 hours e Non migratory We generally use the HT1080 human fibrosarcoma cell line ATCC Catalog no CCL 121 for titering purposes Continued on next page Titering EmGFP Lentivirus Continued Using Polybrene During Transduction Materials Needed Transduction of lentivirus into mammalian cells may be enhanced if cells are transduced in the presence of hexadimethrine bromide Polybrene Note however that some cells are sensitive to Polybrene e g primary neurons Before performing any transduction experiments you may want to test your cell line for sensitivity to Polybrene If your cells are sensitive to Polybrene e g exhibit toxicity or phenotypic changes do not add Polybrene during transduction In this case cells should still be successfully transduced Follow the instructions below to prepare Polybrene Sigma Catalog no H9268 1 Prepare a 6 mg ml stock solution in deioni
23. ents and as a result expression Typically expression levels increase linearly as the MOI increases A number of factors can influence determination of an optimal MOI including the nature of your mammalian cell line e g non dividing vs dividing cell type its transduction efficiency and your application of interest If you are trying to optimize transducing your mammalian cell line of choice for the first time using pLenti6 2 GW EmGFP we recommend using a range of MOIS e g 0 0 05 0 1 0 5 1 2 5 to determine the MOI required to obtain optimal expression of EmGFP in your cell line Continued on next page Transduction and Analysis Continued Concentrating Virus Determining Blasticidin Sensitivity for Your Cell Line Materials Needed Important It is possible to concentrate VSV G pseudotyped lentiviruses using a variety of methods without significantly affecting their transducibility If the titer of your lentiviral stock is relatively low less than 5 x 10 TU ml and your experiment requires that you use a large volume of viral supernatant e g a relatively high MOI you may wish to concentrate your virus before proceeding to transduction For details and guidelines to concentrate your virus refer to published reference sources Yee 1999 If you wish to select for stably transduced cells on your cell line for the first time you must first determine the minimum concentration of Blasticidin required t
24. f 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 Carlsbad California 92008 Phone 760 603 7200 or outlicensing lifetech com Continued on next page 29 Purchaser Notification Continued Limited Use Label License No 267 Mutant Green Fluorescent Products Limited Use Label License No 272 Humanized GFP 30 This product and its use is the subject of one or more of U S Patent Nos 6 090 919 5 804 387 5 994 077 and foreign equivalents This product is the subject of one or more of U S Patent Numbers 5 786 464 5 795 737 5 874 304 and 6 114 148 and foreign equivalents licensed by Life Technologies Corpor ation This product is sold for research use only Not for therapeutic or diagnostic use in humans Gateway Clone Distribution Policy Introduction Gateway Entry Clones Gateway Expression Clones Additional Terms and Conditions The information supplied in this section is intended to provide clarity concerning Invitrogen s policy for the use and distribution of cloned nucleic acid fragments including open reading frames
25. hemically Competent E coli page vi This strain is particularly well suited for use in cloning unstable DNA such as lentiviral DNA containing direct repeats Once you have transformed the Stbl3 E coli select transformants on LB agar plates containing 100 pg ml ampicillin see page 23 for recipe For long term storage we recommend that you make a glycerol stock for long term storage see page 23 Plasmid DNA for transfection into eukaryotic cells must be clean and free from phenol and sodium chloride as contaminants may kill the cells and salt will interfere with lipid complexing decreasing transfection efficiency We recommend isolating plasmid DNA for transfection using the PureLink HiPure Plasmid Midiprep Kit page vi Continued on next page Producing EmGFP Lentivirus in 293FT Cells Continued Determining You may determine transfection efficiency of the pLenti6 2 GW EmGFP Transfection Expression Control Vector into 293FT cells in either of the following ways Efficiency e Qualitatively by examining transfected EmGFP expressing 293FT cells under a fluorescence microscope e Quantitatively by analyzing transfected EmGFP expressing 239FT cells by the flow cytometry method of choice Note If you choose to perform flow cytometry you can use an irrelevant plasmid DNA such as an empty DEST vector instead of the ViraPower Packaging mix to avoid using virus producing cells in your flow cytometer which may present
26. ing Blasticidin Weigh out Blasticidin and prepare solutions in a hood Blasticidin may be obtained separately from Invitrogen page vi in 50 mg aliquots Blasticidin is soluble in water Use sterile water to prepare stock solutions of 5 to 10 mg ml e Dissolve Blasticidin in sterile water and filter sterilize the solution e Aliquot solution in small volumes suitable for one time use see next to last point below 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 2 weeks at 4 C and 6 8 weeks at 20 C e pH of the aqueous solution should be 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 store the thawed stock solution at 4 C for up to 2 weeks Medium containing Blasticidin may be stored at 4 C for up to 2 weeks Map of pLenti6 2 GW EmGFP Expression Control Vector Map of pLenti6 2 The map below shows the elements of the pLenti6 2 GW EmGFP Expression GW EmGFP Control Vector 7883 bp The complete sequence of this vector is available for downloading from our web site at www invitrogen com or by contacting Technical Support page 27 attB1 EmGFP attB2 pLenti6 2 GW EmGFP Comments for pLenti6 2 GW EmGFP 7883 nucleotides RSV enhancer promoter bases 1 229 HIV 1 5 LTR bases 230 410 5 splice donor
27. ing signal Allows viral packaging Luciw 1996 HIV 1 Rev response element RRE Permits Rev dependent nuclear export of unspliced viral mRNA Kjems et al 1991 Malim et al 1989 Human cytomegalovirus CMV immediate early promoter enhancer Allows efficient high level expression of your recombinant protein Andersson et al 1989 Boshart et al 1985 Nelson et al 1987 attB1 and attB2 sites Allow recombination based transfer of EmGFP into any Gateway expression vector via an LR and BP reaction EmGFP Allows visual detection using fluorescence microscopy or flow cytometry PGK Promoter Allows high level expression of Blasticidin in mammalian cell lines EM7 promoter Allows expression of Blasticidin in E coli Blasticidin bsd resistance gene Permits selection of stably transduced mammalian cell lines Kimura et al 1994 SV40 early polyadenylation signal Allows efficient transcription termination and polyadenylation of mRNA Ampicillin bla resistance gene B lactamase Allows selection of transformants in E coli pUC origin Allows high copy number replication and growth in E coli Technical Support Web Resources Visit the Invitrogen web site at www invitrogen com for e Technical resources including manuals vector maps and sequences application notes MSDSs FAQs formulations citations handbooks etc e Complete technical supp
28. ion Protocol 20 Plate cells in complete media as appropriate for your application On the day of transduction Day 1 thaw your lentiviral stock and dilute if necessary the appropriate amount of virus see Determining Optimal MOI page 18 into fresh complete medium Keep the total volume of medium containing virus as low as possible to maximize transduction efficiency Remove the culture medium from the cells Mix the medium containing virus gently by pipetting DO NOT vortex and add to the cells Add Polybrene if desired to the plate a final concentration of 6 pg ml Swirl the plate gently to mix Incubate at 37 C in a CO incubator overnight Note If you are transducing cells with undiluted viral stock and are concerned about possible toxicity or growth effects caused by overnight incubation it is possible to incubate cells for as little as 6 hours prior to changing medium The following day Day 2 remove the medium containing virus and replace with fresh complete culture medium without Blasticidin The following day Day 3 you may analyze the cells for transient expression of EmGFP by flow cytometry see page 15 or by fluorescence microscopy If you wish to select for stably transduced cells continue with Step 8 below Remove the medium and replace with fresh complete medium containing the appropriate amount of Blasticidin as appropriate to select for stably transduced cells see page 19 Replace medium wi
29. ium Add 5 ml of the 293FT cell suspension 6 x 10 total cells to the plate containing media and DNA Lipofectamine 2000 complexes Mix gently by rocking the plate back and forth Incubate cells overnight at 37 C in a CO incubator Protocol continues on next page Continued on next page Producing EmGFP Lentivirus in 293FT Cells Continued 293FT Transfection Protocol Continued Alternate Transfection Protocol Filtering Virus 10 Protocol continued from previous page 8 The next day remove the medium containing the DNA Lipofectamine 2000 complexes and replace with complete culture medium containing sodium pyruvate Return the cells to 37 C in a CO incubator and continue to step 9 next page if you are producing lentivirus Note You may assay for transfection efficiency at 24 48 hours post transfection by fluorescence microscopy see page 6 Greater than 90 of the cells should be EmGFP positive 9 Harvest virus containing supernatants 48 72 hours post transfection by removing medium to a 15 ml sterile capped conical tube Caution Remember that you are working with infectious virus at this stage Follow your institution s guidelines for working with BL 2 organisms 10 Centrifuge the viral supernatant at 3000 rpm for 15 minutes at 4 C to pellet cell debris You can perform a filtration step if desired see below 11 Pipet viral supernatants into cryovials in 1 ml aliquots Store viral stocks at
30. lonies on an LB agar plate containing 100 ug ml ampicillin 2 Isolate a single colony and inoculate into 1 2 ml of LB containing 100 pg ml ampicillin 3 Grow at 37 C with shaking until the culture reaches stationary phase 4 Mix 0 85 ml of culture with 0 15 ml of sterile glycerol 5 Transfer to a cryovial and store at 80 C 23 Blasticidin Blasticidin Molecular Weight Formula and Structure Handling Blasticidin Preparing and Storing Stock Solutions 24 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 nontoxic deaminohydroxy derivative Izumi et al 1991 Blasticidin is available separately from Invitrogen see page vi for ordering information For information on preparing and handling Blasticidin see the Appendix page 24 The formula for Blasticidin S is Ci7HzsNsOs HCL and the molecular weight is 458 9 The diagram below shows the structure of Blasticidin NH2 Sn ee HOOC o CH3 HCl adds d NH NH Always wear gloves mask goggles and protective clothing e g a laboratory coat when handl
31. ly CMV promoter enhancer for high level expression of EmGFP in a wide range of mammalian cells e Emerald Green Fluorescent Protein EmGFP derived from Aequorea victoria GFP for fluorescence detection e Murine PGK promoter for high level expression of the Blasticidin resistance gene e Blasticidin resistance gene for selection in E coli and mammalian cells Izumi et al 1991 Kimura et al 1994 Takeuchi et al 1958 Yamaguchi et al 1965 e Ampicillin resistance gene for selection in E coli e pUC origin for high copy replication of the plasmid in E coli For the map and features of the pLenti6 2 GW EmGFP Expression Control Vector see page 25 Continued on next page Overview Continued Important Components of the ViraPower Lentiviral Expression System pLenti6 2 GW EmGFP To produce lentivirus using the pLenti6 2 GW EmGFP Expression Control Vector you must supply the components of the ViraPower or ViraPower II Lentiviral Expression System including 293FT cells Lipofectamine 2000 Opti MEM I and the ViraPower Packaging Mix These components are described in the section below Ordering information for these products can be found on page vi TM The ViraPower Lentiviral Expression System facilitates highly efficient in vitro or in vivo delivery of a target gene to dividing and non dividing mammalian cells using a replication incompetent lentivirus Based on the lentikat system develo
32. nate transfection protocol page 10 Transfected cells not cultured in One day after transfection remove media media containing sodium pyruvate 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 Viral supernatants can generally be early collected 48 72 hours post transfection 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 Viral supernatant too dilute Concentrate virus using any method of choice Yee 1999 Viral supernatant frozen and Do not freeze thaw viral supernatant thawed multiple times more than 3 times Poor choice of titering cell line Use HT1080 cells or another adherent cell line with the characteristics discussed on page 12 Continued on next page 21 Troubleshooting Continued Problem Cause Solution Low viral titer continued Polybrene not included during transduction Transduce pLenti6 2 GW EmGFP into cells in the presence of Polybrene Lipofectamine 2000 handled incorrectly e Store at 4 C Do not freeze e Mix gently by inversion before use Do not vortex No EmGFP positive cells obtained after titering Incorrect filter set on cell culture fluorescence microscope or detection parameters for flow cytomete
33. ng 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 31 References Andersson S Davis D L Dahlback H J rnvall H and Russell D W 1989 Cloning Structure and Expression of the Mitochondrial Cytochrome P 450 Sterol 26 Hydroxylase a Bile Acid Biosynthetic Enzyme J Biol Chem 264 8222 8229 Boshart M Weber F Jahn G Dorsch H sler K Fleckenstein B and Schaffner W 1985 A Very Strong Enhancer is Located Upstream of an Immediate Early Gene of Human Cytomegalovirus Cell 41 521 530 Ciccarone V Chu Y Schifferli K Pichet J P Hawley Nelson P Evans K Roy L and Bennett S 1999 Lipofectamine 2000 Reagent for Rapid Efficient Transfection of Eukaryotic Cells Focus 21 54 55 Dull T Zufferey R Kelly M Mandel R J Nguyen M Trono D and Naldini L 1998 A Third Generation Lentivirus Vector with a Conditional Packaging System J Virol 72 8463 8471 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 Bio
34. o kill your untransduced mammalian cell line i e perform a kill curve experiment See the ViraPower System Manual for more information about determining Blasticidin sensitivity See page 24 for information on handling and storing Blasticidin You will need the following items e Your titered lentiviral stock stored at 80 C until use e Mammalian cell line of choice e Complete culture medium for your cell line e 6mg ml Polybrene see page 13 e Appropriately sized tissue culture plates for your application e Inverted fluorescence microscope with FITC filter or Omega XF100 filter see page 7 for detecting EmGFP expressing cells in culture or a flow cytometry system with a FITC filter to quantitatively detect EmGFP expressing cells e Blasticidin if selecting for stably transduced cells Remember that viral supernatants are generated by harvesting spent media containing virus from the 293FT producer cells Spent media lacks nutrients and may contain some toxic metabolic waste products If you are using a large volume of viral supernatant to transduce your mammalian cell line e g 1 ml of viral supernatant per well in a 6 well plate note that growth characteristics or morphology of the cells may be affected during transduction These effects are generally alleviated after transduction when the media is replaced with fresh complete media Continued on next page 19 Transduction and Analysis Continued Transduct
35. 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 In the following example an EmGFP lentiviral stock was generated using the protocol on the previous page The stock was concentrated and the following data were generated after performing flow cytometry Lentivirus Dilution EmGFP Positive Cells 10 91 5 10 34 6 104 44 The following formula White et al 1999 Sastry et al 2002 is used to calculate the titer F x C V x D F frequency of GFP positive cells percentage obtained divided by 100 C total number of cells in the well at the time of transduction V volume of inoculum in ml D lentivirus dilution In the above example the 10 dilution is used to calculate the titer since the percentage of EmGFP positive cells falls into the desired range of 1 30 The frequency of EmGFP positive cells is 4 4 100 0 044 multiplied by 2 x 10 the number of cells in the well divided by 1 the volume of inoculum Thus the calculation is as follows 0 044 x 200 000 1 x 10 The titer for this example is 8 8 x 107 TU ml We typically obtain unconcentrated EmGFP lentivirus titers in the range of 5 x 105 2 x 10 T
36. of the following and mix gently To Generate Lentivirus from To Check 293FT Transfection 3 ug of pLenti6 2 GW EmGFP 1 5 ml of Opti MEM I Medium without serum 293FT Cells combine Efficiency without Generating Lentivirus combine 9 ug of the ViraPower 9 ug of an irrelevant plasmid Packaging Mix DNA see page 6 3 ug of pLenti6 2 GW EmGFP 1 5 ml of Opti MEM I Medium without serum TM 2 Inaseparate sterile 15 ml tube mix Lipofectamine 2000 gently before use then dilute 36 ul of Lipofectamine 2000 in 1 5 ml of Opti MEM I Medium without serum Mix gently and incubate for 5 minutes at room temperature After the 5 minute incubation combine the diluted DNA from Step 1 with the diluted Lipofectamine 2000 from Step 2 Mix gently Incubate for 20 minutes at room temperature to allow the DNA TM Lipofectamine 2000 complexes to form The solution may appear cloudy but this will not impede the transfection While DNA lipid complexes are forming trypsinize and count the 293FT cells Resuspend the cells at a density of 1 2 x 10 cells ml in growth medium or Opti MEM I Medium containing FBS at the same concentration as the growth medium for that cell line Do not include antibiotics in the medium Add the DNA Lipofectamine 2000 complexes to a 10 cm tissue culture plate containing 5 ml of growth medium or Opti MEM I Medium containing serum Do not include antibiotics in the med
37. operties of Aequorin a Bioluminescent Protein from the Luminous hHydromedusan Aequorea Journal of Cellular and Comparative Physiology 59 223 239 Takeuchi S Hirayama K Ueda K Sakai H and Yonehara H 1958 Blasticidin S A New Antibiotic The Journal of Antibiotics Series A 11 1 5 Tsien R Y 1998 The Green Fluorescent Protein Annu Rev Biochem 67 509 544 White S M Renda M Nam N Y Klimatcheva E Y Zhu Fisk J Halterman M Rimel B J Federoff H Pandya S Rosenblatt J D and Planelles V 1999 Lentivirus vectors using human and simian imunodeficiency virus elements J Virology 73 2832 2840 Yamaguchi H Yamamoto C and Tanaka N 1965 Inhibition of Protein Synthesis by Blasticidin S I Studies with Cell free Systems from Bacterial and Mammalian Cells J Biochem Tokyo 57 667 677 Yee J K 1999 in The Development of Human Gene Therapy Friedmann T ed pp 21 45 Cold Spring Harbor Laboratory Press Cold Spring Harbor NY Zhang G Gurtu V and Kain S 1996 An Enhanced Green Fluorescent Protein Allows Sensitive Detection of Gene Transfer in Mammalian Cells Biochem Biophys Res Comm 227 707 711 2005 2008 2010 Invitrogen Corporation All rights reserved For research use only Not intended for any animal or human therapeutic or diagnostic use 32 invitrogen Corporate Headquarters Invitrogen Corporation 5791 Van Allen Way Carlsbad CA 92008 T 1 760
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39. otein GFP GFP and Spectral Variants This manual provides instructions and guidelines to 1 Transfect pLenti6 2 GW EmGFP Expression Control Vector into the 293FT Cell Line to determine transfection efficiency 2 Co transfect the pLenti6 2 GW EmGFP Expression Control Vector and the TM ViraPower Packaging Mix into the 293FT Cell Line to produce a control lentiviral stock 3 Titer the control lentiviral stock using fluorescence detection methods 4 Usethe control lentiviral stock to determine optimal transduction conditions for your mammalian cell line of choice TM For more information about the ViraPower or ViraPower II System refer to the ViraPower System Manual For instructions to culture and maintain the 293FT producer cell line refer to the 293FT Cell Line manual These manuals are supplied with the ViraPower Lentiviral Expression Kits and are also available for downloading from www invitrogen com or by contacting Technical Support page 27 Green Fluorescent Protein GFP is a naturally occurring bioluminescent protein derived from the jellyfish Aequorea victoria Shimomura et al 1962 GFP emits fluorescence upon excitation and the gene encoding GFP contains all of the necessary information for posttranslational synthesis of the luminescent protein GFP is often used as a molecular beacon because it requires no species specific cofactors for function and the fluorescence is easily detected u
40. otein so that mutations appear to be increased by one position For example the S65T mutation actually occurs in codon 66 of EmGFP The EmGFP expressed from the pLenti6 2 GW EmGFP Expression Control Vector has the following excitation and emission wavelengths as published in the literature Tsien 1998 Fluorescent Protein Excitation nm Emission nm EmGFP 487 509 The fluorescent signal from EmGFP can be detected with standard FITC filter sets for fluorescence microscopy systems However for optimal detection of the fluorescent signal you may use a filter set which is optimized for detection within the excitation and emission ranges for each of the fluorescent proteins This filter set and the manufacturer is listed below Fluorescent Filter Set for Protein Fluorescence Manufacturer Microscopy EmGFP Omega XF100 Omega www omegafilters com For information on obtaining this filter set contact Omega Optical Inc www omegafilters com Methods Producing EmGFP Lentivirus in 293FT Cells Introduction Using the Vector Propagating the Vector Plasmid DNA You can use the Vivid Colors pLenti6 2 GW EmGFP Expression Control Vector to transfect 293FT cells estimate the transfection efficiency and produce EmGFP lentiviral stocks The following section provides guidelines and protocols for performing these steps Lentivirus produced with the ViraPower System can
41. ped by Cell Genesys Dull et al 1998 the ViraPower Lentiviral Expression System possesses features which enhance its biosafety while allowing high level gene expression in a wider range of cell types than traditional retroviral systems The System includes the following major components e ApLenti based expression vector such as pLenti6 2 GW EmGFP that contains the elements required for packaging into virions e g 5 and 3 LTRs Y packaging signal e The ViraPower Packaging Mix that contains an optimized mixture of the three packaging plasmids pLP1 pLP2 and pLP VSVG These plasmids supply the helper functions as well as structural and replication proteins in trans required to produce the lentivirus e The 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 The pLenti6 2 GW EmGFP Expression Control Vector was generated by performing an LR recombination reaction between an entry vector containing the EmGFP gene and the pLenti6 2 V5 DEST vector The attB sites flanking EmGFP are a result of the LR recombination reaction Note There is a V5 epitope from the pDEST vector backbone downstream of the EmGFP gene but it will NOT be expressed due to a stop codon at the end of the EmGFP coding sequence Continued on next page Overview Continued Purpose of the Manual Additional Information Green Fluorescent Pr
42. pplications 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 one or more of U S Patent Nos 5 491 084 and 6 146 826 and foreign equivalents 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 This product and its use is the subject of one or more of U S Patent Nos 5 777 079 6 066 476 and 6 319 669 and foreign equivalents 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 produ
43. pport Representatives Invitrogen warrants that all of its products will perform according to specifications stated on the certificate of analysis The company will replace free of charge any product that does not meet those specifications This warranty limits Invitrogen Corporation s liability only to the cost of the product No warranty is granted for products beyond their listed expiration date No warranty is applicable unless all product components are stored in accordance with instructions Invitrogen reserves the right to select the method s used to analyze a product unless Invitrogen agrees to a specified method in writing prior to acceptance of the order Invitrogen makes every effort to ensure the accuracy of its publications but realizes that the occasional typographical or other error is inevitable Therefore Invitrogen makes no warranty of any kind regarding the contents of any publications or documentation If you discover an error in any of our publications please report it to our Technical Support Representatives Invitrogen assumes no responsibility or liability for any special incidental indirect or consequential loss or damage whatsoever The above limited warranty is sole and exclusive No other warranty is made whether expressed or implied including any warranty of merchantability or fitness for a particular purpose 27 Purchaser Notification Introduction Limited Use Label License No 19 Gateway
44. r Make sure you are using a FITC or Omega XF100 filter set on your inverted fluorescence microscope see page 7 or the FITC detection parameters on your flow cytometer Viral stocks stored incorrectly Aliquot and store stocks in cryovials at 80 C Do not freeze thaw more than 3 times Polybrene not included during transduction Transduce pLenti6 2 GW EmGFP into cells in the presence of Polybrene Too soon to see EmGFP expression For optimal EmGFP expression wait 4 days post transduction Poor expression of EmGFP in transiently transduced mammalian cell lines Low transduction efficiency e Polybrene not included during transduction e Non dividing cell type used e Transduce pLenti6 2 GW EmGFP into cells in the presence of Polybrene e Transduce pLenti6 2 GW EmGFP into cells using a higher MOI MOI too low Transduce pLenti6 2 GW EmGFP into cells using a higher MOI Cells harvested too soon after transduction Do not harvest cells until at least 48 72 hours after transduction to allow EmGFP to accumulate in transduced cells No expression of EmGFP after stable transduction into mammalian cell lines Promoter silencing pLenti6 2 GW EmGFP may integrate into a chromosomal region that silences the CMV promoter controlling expression ofEmGFP Screen multiple antibiotic resistant clones and select the one with the highest expression levels Incorrect filte
45. r used with cell culture fluorescence microscope or detection parameters for flow cytometer Make sure you are using a FITC or Omega XF100 filter set on your inverted fluorescence microscope see page 7 or the FITC detection parameters on your flow cytometer Poor expression of EmGFP after stable transduction into mammalian cell lines Too much Blasticidin 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 22 Recipes LB Luria Bertani Medium and Plates Making Glycerol Stocks for Long Term Storage Appendix Composition 1 0 Tryptone 0 5 Yeast Extract 1 0 NaCl pH 7 0 1 For 1 liter dissolve 10 g tryptone 5 g yeast extract and 10 g NaCl in 950 ml deionized water 2 Adjust the pH of the solution to 7 0 with NaOH and bring the volume up to 1 liter 3 Autoclave on liquid cycle for 20 minutes at 15 psi Allow solution to cool to 55 C and add antibiotic if needed 4 Store at room temperature or at 4 C For LB agar plates 1 Prepare LB medium as above but add 15 g L agar before autoclaving 2 Autoclave on liquid cycle for 20 minutes at 15 psi 3 After autoclaving cool to 55 C add antibiotic if needed and pour into 10 cm plates 4 Letharden then invert and store at 4 C 1 Streak the original colony out to obtain single co
46. reat used pipets 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 Continued on next page 13 Titering EmGFP Lentivirus Continued Transduction Procedure 14 Follow the procedure below to determine the titer of your lentiviral stock using the mammalian cell line of choice You will use at least one 6 well plate for each lentiviral stock to be titered usually one mock well plus five dilutions 1 24 hours before transduction Day 1 trypsinize and count the cells plating them in a 6 well plate such that they will be 25 confluent at the time of transduction For example when using HT1080 cells plate 1 x 10 cells per well of a 6 well plate Incubate cells at 37 C in a CO incubator overnight Alternate plating method for HT1080 cells The morning of transduction Day 2 plate 2 x 10 cells well in a 6 well plate In the afternoon after cells have adhered to the plate approximately 4 5 hours transduce as described below On the day of transduction Day 2 thaw your lentiviral stock In a biosafety cabinet prepare five 10 fold serial dilutions ranging from 10 to 10 You should also prepare a mock dilution containing no virus For each dilution dilute the lentiviral stock into complete culture medium containing 6 8 ug ml Polybrene optional se
47. sing fluorescence microscopy and standard filter sets Modifications have been made to the wild type GFP to enhance its expression in mammalian systems These modifications include nucleic acid substitutions that correspond to the codon preference for mammalian use and mutations that increase the brightness of the fluorescence signal resulting in enhanced GFP Zhang et al 1996 Mutations have also arisen or have been introduced into GFP that further enhance and shift the spectral properties of GFP such that these proteins will emit fluorescent color variations reviewed in Tsien 1998 The Emerald GFP EmGFP is a variant of enhanced GFP Continued on next page Overview Continued EmGFP Spectral Properties of EmGFP Fluorescence Filter Set for Detecting EmGFP Fluorescence The EmGFP variant has been described in a published review Tsien 1998 and the amino acid changes are summarized in the table below The mutations are represented by the single letter abbreviation for the amino acid in the consensus GFP sequence followed by the codon number and the single letter amino acid abbreviation for the substituted amino acid Fluorescent Protein GFP Mutations EmGFP S65T S72A N149K M153T 1167T Mutations listed are as described in the literature When examining the actual sequence the vector codon numbering starts at the first amino acid after the initiation methionine of the fluorescent pr
48. ssing cells Continued on next page Producing EmGFP Lentivirus in 293FT Cells Continued 293FT Cell Line E N NOD e NN o 2 E Detecting EmGFP by Fluorescence Microscopy The human 293FT Cell Line is optimized for 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 TM The 293FT Cell Line is supplied with the ViraPower Lentiviral Expression kits and is also available separately from Invitrogen page vi For more information about how to culture and maintain 293FT cells refer to the 293FT Cell Line manual This manual is supplied with the ViraPower Lentiviral Expression kits and is also available for downloading from www invitrogen com or by contacting Technical Support page 27 The health of your 293FT cells at the time of transfection has a critical effect on the success of lentivirus production Use of unhealthy cells 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 healthy and greater than 90 viable e Subculture and maintain cells as recommended in the 293FT
49. t 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 infringe ment of the above patents based upon the use of this product to manufacture a protein for sale provided that no method claim in the above patents was used in the manufacture of such protein If the purchaser is not willing to accept the limitations of this limited use statement 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 For additional information about Invitrogen s policy for the use and distribution of Gateway clones see the section entitled Gateway Clone Distribution Policy page 31 Blasticidin and the blasticidin resistance gene bsd are the subject of U S Patent No 5 527 701 sold under patent license for research purposes only For information on purchasing a license to this product for purposes other than research contact Licensing Department Life Technologies Corpor ation 5791 Van Allen Way Carlsbad California 92008 Phone 76
50. th fresh medium containing antibiotic every 3 4 days until antibiotic resistant colonies can be identified generally 10 12 days after selection Pick at least 5 antibiotic resistant colonies and expand each clone to analyze the expression of EmGFP by flow cytometry see page 15 or by fluorescence microscopy Troubleshooting Introduction The table below lists some potential problems and solutions that may help you troubleshoot EmGFP lentivirus production titering and transduction of cells with EmGFP lentivirus Problem Cause Solution Low viral titer Low transfection efficiency e Used poor quality plasmid DNA e Do not use mini prep plasmid DNA Le plasmid DNA from a mini for transfection Use the PureLink prep Midiprep Isolation Kit page vi or CsCl gradient centrifugation to prepare plasmid DNA e Unhealthy 293FT cells cells e Use healthy 293FT cells under exhibit low viability passage 20 do not overgrow e Cells transfected in media e Although Geneticin is required for containing antibiotics i e stable maintenance of 293FT cells Do Geneticin not add Geneticin to media during transfection as this reduces transfection efficiency and causes cell death e Plasmid DNA transfection e Usea DNA in ug Lipofectamine reagent ratio incorrect 2000 in pl ratio ranging from 1 2 to 1 3 e 293FT cells plated too sparsely e Plate cells as recommended in the transfection protocol page 9 or try the alter
51. you are ready to determine the titer of your viral stock Since cells that are transduced with EmGFP lentivirus produce EmGFP the titer of the EmGFP lentivirus stock can be calculated at 4 days post transduction by fluorescence detection and without the need for lengthy antibiotic selection Protocols and guidelines are provided in this section to titer your EmGFP lentiviral stock To determine the titer of your EmGFP lentiviral stock you will 1 Prepare 10 fold serial dilutions of your EmGFP lentiviral stock 2 Transduce the different dilutions of lentivirus into cells in the presence of Polybrene 3 Determine the lentiviral titer by fluorescence detection at 4 days post transduction The pLenti6 2 GW EmGFP Expression Control Vector contains the Blasticidin resistance gene You may use the standard titer method based on antibiotic selection described in the ViraPower System manual If you are generating lentivirus for the first time and do not know what to expect you may wish to generate a wider range i e 10 10 of dilutions in the event that your virus stock turns out to have a very high or very low titer e If calculating the most accurate titer is critical for your experiments you may wish to set up triplicate transductions for each dilution and use the average percentage of GFP positive cells for your calculations You may titer your lentiviral stock using any mammalian cell line of choice Generally we r
52. zed sterile water 2 Filter sterilize and dispense 1 ml aliquots into sterile tubes 3 Store at 20 C for long term storage Stock solutions may be stored at 20 C for up to 1 year Do not freeze thaw the stock solution more than 3 times as this may result in loss of activity Note The working stock of Polybrene may be stored at 4 C for up to 2 weeks Polybrene is a registered trademark of Abbott Laboratories You will need the following items e Your EmGFP lentiviral stock store at 80 C until use e Adherent mammalian cell line of choice see page 12 e Complete culture medium for your cell line e Optional 6 mg ml Polybrene see above e 6 well tissue culture plates e Inverted fluorescence microscope with FITC filter or Omega XF100 filter see page 7 for detecting EmGFP expressing cells in culture or a flow cytometry system with a FITC filter to quantitatively detect EmGFP expressing cells e Optional Trypsin or cell dissociation solution of choice if performing flow cytometry e Optional Flow cytometry buffer of choice such as calcium magnesium free Phosphate Buffered Saline containing 1 FBS or BSA if performing flow cytometry Remember that you will be working with media containing infectious virus Follow the recommended Federal and institutional guidelines for working with BL 2 organisms e Perform all manipulations within a certified biosafety cabinet e Treat media containing virus with bleach e T
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