Cellecta-Manual-Lenti
Contents
1. 0 0 5 1 1 5 2 2 5 MOI viral particles cell The of infected cells is determined by flow cytometry excitation 56inm emission 600 20 for TagRFP by observing the of RFP cells in the transduced cell sample When the of infected cells is at or below 20 the of integrations is with good approximation equivalent to the of transduced cells At higher transduction efficiencies the fraction of transduced cells bearing multiple integrations becomes higher and higher so that the increase in of transduced cells relative to integration events cell is no longer linear Using the table below MOI MOI multiplicity of infection viral particles cell can be calculated with good accuracy in the range 0 2 1 5 MOI For individual constructs MOI can also be calculated higher than 2 5 in order to achieve gt 90 transduction efficiency Titer is calculated according to the TITER FORMULA below TU MOI llsat T ducti ml Graf cells t Transduction ml of Viral Stock used at Transduction Example IF The original of cells at Transduction was 100 000 and The volume of virus stock used was 10 ul and The observed of transduced RFP cells is 25 THEN The calculated MOI is 0 3 from the chart and The TITER is 100 000 93 i 0 01 3 000 000 TU ml tech cellecta com 11 of 21 v9 5 10 2015 Lentiviral Packaging Titering and Transduction www cellecta com Use2. Using blue laser 488nm for excitation leads to gross underestimation of viral titer Problem Inthe RFP assay the of transduced cells is determined by fluorescence microscopy instead of flow cytometry Solution Use flow cytometry H 3 Transduction affects target cell viability Problem Polybrene is toxic for target cells Solution Optimize the concentration and exposure time to Polybrene during the transduction step For some sensitive cells Polybrene should not be used Problem Virus containing conditioned media is toxic to target cells Solution Concentrate and resuspend the virus in target cell growth media PBS 10 FBS or PBS 1 BSA H 4 No expression of RFP or Puro or shRNAs in target cells Problem The promoter is not functional in target cells Solutions e Change the target cells e Contact Cellecta at tech cellecta com to have the library cloned in another vector with different promoter tech cellecta com 17 of 21 v9 5 10 2015 Lentiviral Packaging Titering and Transduction User Manual I Technical Support www cellecta com Please let us know the nature of the problem you are having by answering the following questions Packaging What was the lentiviral titer and what was the total number of TU packaged How was the virus concentrated if applicable Transducing Target Cells What MOI did you use to transduce your target cells What target cells did you use H
3. e Fetal Bovine Serum recommended Mediatech Cat MT 35 010 CV e Puromycin e D PBS Mediatech Cat 21 031 CV e Trypsin EDTA Mediatech Cat 15 040 CV e Polybrene hexadimethrine bromide Sigma Aldrich Cat 107689 e LentiFuge lentiviral concentration reagent Cellecta e 500 ml 0 2 um filter units Fisher Scientific Cat 09 741 05 or Thermo Scientific Cat A 569 0020 e Tissue Culture Plates and Related Tissue Culture Supplies e Lipofectamine Reagent Life Technologies Cat 18324 020 e Plus Reagent Life Technologies Cat 11514 015 C Recommended Pilot Experiments C 1 Doubling Time The doubling time is the time it takes your model cells to double in number It is useful to know the doubling time of your cells so that you can plate the appropriate number for transduction with the lentiviral library Start with cells that have already been growing for a few weeks rather than using cells that have just been thawed from a frozen state To calculate the doubling time trypsinize your cells as if you were going to split them Count them using a hemacytometer or cell counter and keep track of the number that you replate onto the cell culture plates The starting number of cells is Xb Propagate the cells as you normally do replacing media as necessary The next time they are ready to be split trypsinize them as usual and count them again using a hemacytometer or cell counter The number of cells at the end is ref
4. including lentiviral vector concentration exposure time to lentiviral vector and growth area of cells To determine the concentration of lentiviral vector particles required to provide the desired multiplicity of infection MOI for your target cells you should titer the viral stock in the same target cells used in the experiment e Cellecta s Lentiviral vectors contain a deletion in the 3 LTR that leads to self inactivation of the lentiviral vector after reverse transcription and integration into genomic DNA Although more than one copy of a lentiviral construct may be integrated into the genome of a single cell the lentiviral construct cannot produce infectious viral particles However despite these safety features please remember that you are working with transducible lentiviral vector particles Although the particles are replication incompetent they are infection competent so the expression cassette that they carry can potentially infect integrate and express in any mammalian cell Please follow the recommended guidelines for working with Biosafety Level 2 BSL 2 materials tech cellecta com 13 of 21 v9 5 10 2015 Lentiviral Packaging Titering and Transduction www cellecta com User Manual G 2 Transduction Protocol HEK293 Easy to Transduce Cells Please read the entire protocol before beginning your experiment For other plate formats and other cell types the volumes cell number and media should be adjusted dependi
5. 5 ug ml is recommended For some cell types you cannot use Polybrene CA Promoter Validation If you have not used lentiviral vectors in your target cells before you may wish to do a pilot experiment to determine which promoters will work best Most vectors have a cDNA promoter for expression of the RFP and Puro resistance as well as a shRNA promoter for expression of the shRNA Cellecta sells pre packaged viruses expressing different marker genes from different promoters You can use these to determine which promoter combination will work the best for your cells For more information please see http www cellecta com products and services pooled lentiviral libraries control shRNA constructs D Packaging Protocol for Individual Lentiviral Constructs The following protocol describes the generation of pseudoviral packaged lentiviral constructs using Invitrogen s Lipofectamine and Plus Reagent see Required Materials Other transfection reagents may be used but the protocol should be adjusted to fit the manufacturer s protocol The yield of recombinant lentiviral particles typically produced under these optimized conditions is 1 x 10 TU ml for shRNA constructs CRISPR constructs or cDNA constructs may have a lower yield depending on the size of the lentiviral construct This protocol uses 10 cm plates Multiple plates can be used and viral supernatant combined to increase the yield of virus tech cellecta com 5 of 21 v9 5
6. Portions of the Product are covered by US and foreign patent applications or patents and other proprietary intellectual property rights owned by CSHL shRNA IP Rights Subject to Acceptance and all terms and conditions of this License sale of the Product to Customer by Seller acting under its license from CSHL an Authorized Sale conveys to Customer only the nonexclusive nontransferable right under the shRNA IP Rights to use the Product solely for Customer s internal research purposes and only at its facility where the Products are delivered by Seller tech cellecta com 20 of 21 v9 5 10 2015 Lentiviral Packaging Titering and Transduction www cellecta com User Manual Unlicensed Products Any Product that is acquired other than pursuant to an Authorized Sale including without limitation any Product not acquired from Seller shall be deemed to be an Unlicensed Product This License shall be void and of no effect for Unlicensed Products and shall not convey any express or implied right to make use or sell Unlicensed Products for any purpose Restrictions Customer obtains no right to sublicense it rights or to use the Product for the benefit of any third party for any commercial purpose including without limitation using the Product in connection with providing services to any third party or generating commercial databases The Product may not be used in vitro or in vivo for any diagnostic preventative therapeutic or
7. can now be analyzed using an appropriate assay If prolonged shRNA expression is required it is recommend that cells are kept under optimal growth conditions until harvesting H Troubleshooting H 1 Low Lentiviral Titer lt 10 TU ml in supernatant H 1 1 Poor transfection efficiency 48 hour post transfection less than 80 of 293T cells are very brightly fluorescent Problem 293T Cells have too high or too low density Solution Plate fewer or more cells in order to have about 80 confluency at time of transfection Problem Plasmid DNA Lipofectamine Plus Reagent ratios are incorrect Solution Optimize the ratios using the guidelines provided in the Lipofectamine protocol H 1 2 Inefficient production of the virus Problem 293T Cells are of poor quality Solutions e Optimize growth conditions check growth medium and don t grow 293T cells for more than 20 passages e Check for mycoplasma contamination tech cellecta com 15 of 21 v9 5 10 2015 Lentiviral Packaging Titering and Transduction www cellecta com User Manual e Do not overgrow the cells do not allow the cells to reach more than 90 confluency in order to keep the culture continuously in logarithmic growth phase Problem Lentiviral supernatant harvested too early or too late Solution Harvest supernatant 48 hours and 72 hours after transfection Problem 293T cell media is too acidic at time of virus harvesting Solution Make sure to replace me
8. different of RFP cells will express enough Puro to survive puromycin selection i e the higher the MOI the higher the of multiple integrants so the higher the of RFP cells expressing higher levels of Puro In order to calculate which fraction of RFP cells are going to survive puromycin selection the following procedure is strongly suggested Titer virus in target cell line by flow cytometry F 2 Lentiviral Titer estimation RFP assay Based on assessed titer perform a small scale transduction aiming at 50 infected cells 3 days after transduction split cells into 2 samples grow cells puromycin for an additional 3 days then analyze both samples by flow cytometry By looking at the RFP intensity of puromycin treated cells calculate the of cells that survived puromycin selection The figure below shows FACS analysis of transduced cells no puromycin selection blue puromycin selection red 50 of cells were RFP 24 of the RFP cells were also puromycin resistant 12 of total tech cellecta com 12 of 21 v9 5 10 2015 Lentiviral Packaging Titering and Transduction www cellecta com User Manual ample Name IMPORTANT The of RFP cells that are also puromycin resistant is dependent on MOI as it Ess nD M1 253 ks increases with the increase of RFP cells bearing multiple integrations In the example above 24 of RFP cells 12 of total were puromycin resistant when cells were infected at M
9. end of centrifugation you may or may not be able to see a pellet assume it is at the location of the mark 4 Immediately discard the supernatant by aspirating Place the tubes on ice resuspend the in visible pellet in PBS or PBS 10 FBS or PBS 1 BSA make aliquots and freeze at 80 C Alternatively you may concentrate virus by any of the methods below However the yield of virus is superior 80 recovery using Cellecta s protocol above e Ultracentrifugation at 50 000 x g for 90 minutes at 4 C e Sucrose cushion ultracentrifugation tech cellecta com 8 of 21 v9 5 10 2015 Lentiviral Packaging Titering and Transduction www cellecta com User Manual e PEG precipitation followed by centrifugation F Transduction Lentiviral Titer Estimation The following section uses packaged lentiviral particles for transduction into example target cells HEK293 Please note that lentiviral particles should only be opened within the laminar flow hood and should be used under biosafety Level 2 conditions For more information on the biosafety of lentiviral particles please refer to J Safety Guidelines below F 1 Transduction for Lentiviral Titering Lentiviral transductions are performed by mixing cells and virus in culture media supplemented with LentiFuge For both adherent and suspension cells transductions are initiated in suspension and carried out overnight Adherent cells are allowed to adhere to substrate during
10. loan lease rental or any other means to any commercial partner or any other third party for any commercial purpose except only in the following case Customer may transfer the unmodified Product to a commercial third party contractor who pursuant to a written agreement with Customer and only for non royalty based payment s undertakes on behalf of Customer to use the Product solely for Customer s benefit and internal research purposes which third party shall not after termination of such work retain or receive subsequent rights to possess access or use any Product or any results of such work and from whom Customer receives no payments pursuant to such agreement Compliance Customer may only use the Product in compliance with all local state federal and other applicable laws regulations and rules including without limitation for uses in the United States EPA FDA USDA and NIH guidelines Customer may not directly or indirectly use the Product or allow the transfer transmission export or re export of all or any part of the Product or any product thereof in violation of any export control law or regulation of the United Sates or any other relevant jurisdiction Disclaimers THE PRODUCT IS PROVIDED AS IS WITHOUT WARRANTY OF ANY KIND NO WARRANTY IS MADE THAT THE PRODUCT WILL MEET CUSTOMER S REQUIREMENTS OR THAT ANY RESULT CAN BE ACHIEVED OR THAT USE OF THE PRODUCT WILL NOT INFRINGE ANY PATENT OR OTHER PROPRIETARY RIGHT AL
11. transduction and are transduced at a cell density that allows for 2 3 population doublings before reaching confluence Suspension cells are typically transduced at higher density than standard growth density and then they are diluted to standard growth density 18 24 hours after transduction F 1 1 Transduction of Adherent Cells HEK293 cells Titering The following protocol has been optimized for HEK293 cells For other adherent cell types parameters such as media growth surface time of detection etc will have to be adjusted Day 1 1 Quickly thaw the lentiviral particles in a water bath at 37 C Transfer the thawed particles to a laminar flow hood gently mix by rotation inversion or gentle vortexing and keep on ice CAUTION Only open the tube containing the lentiviral particles in the laminar flow hood NOTE Unused lentiviral stock may be refrozen at 80 C but it will typically result in a loss of about 20 in titer 2 Trypsinize and resuspend HEK293 cells to a density of 1 x 10 cells ml in D MEM supplemented with 10 FBS and 5 ug ml Polybrene Aliquot 1 ml well in a 12 well plate and add O ul 3 ul 10 ul 33 ul and 100 ul of lentiviral stock supernatant filtered to remove cells and cell debris not concentrated to six different wells If concentrated virus is used scale down virus volumes accordingly Mix and return cells to CO2 incubator Grow cells under standard conditions for 24 hours NOTE It is important
12. vaccine application or used directly or indirectly in humans for any purpose Customer may not isolate extract reverse engineer derive copy or separately use any component of the Product such as for example any shRNA component for any commercial purpose including without limitation for the purpose of making Products other than solely for Customer s internal research purposes Non Profit Customers If Customer is a Non Profit Entity then the following additional restrictions shall apply Customer obtains no right to use the Product for any commercial purpose Commercial Customers If Customer is a Commercial Entity unless Customer has already entered into a separate written agreement that has been executed by CSHL that covers the shRNA IP rights and that is then currently in effect then the following additional restrictions shall apply This License and Customer s rights hereunder automatically terminate 1 year after delivery of Product to Customer After 1 year of Product use customer must enter into a separate written agreement with CSHL that covers the shRNA IP rights or Customer shall immediately stop using and destroy all Product in its possession The Product may not be used to make any mouse that is of a strain of mice for germ line transmission by embryonic transfer of a gene encoding an shRNA that induces suppression of a gene or genes by RNAi No Transfers Customer may not distribute or transfer the Product by license sale
13. 10 2015 Lentiviral Packaging Titering and Transduction www cellecta com User Manual This protocol can be used to package lentiviral vectors expressing shRNA CRISPR cDNA or promoter reporters 1 Start growing 293T cells in D MEM medium plus glutamine see Required Materials supplemented with 10 FBS without antibiotics 2 to 3 days prior to transfection D 1 Day O Plate Cells 2 Twenty four 24 hours prior to transfection plate 5 5 x 106 293T cells in 10 cm plates or 55 cm flasks Use 10 ml of media per plate Disperse the cells and ensure even distribution At the moment of transfection the cells should have reached 80 confluency Increase or decrease the number of 293T cells seeded if optimal confluency is not achieved in 24 hours Incubate at 37 C in a CO2 incubator for 24 hours The goal is that the 293T cells should reach 80 confluency by Day 1 You may want to calculate the number of seed cells empirically since cell counts can vary tech cellecta com 6 of 21 v9 5 10 2015 Lentiviral Packaging Titering and Transduction www cellecta com User Manual D 2 Day 1 Transfection ten 10 cm plates 3 4 Make a master mix by combining 30 ul of Lipofectamine Reagent and 1 ml of D MEM medium without serum or antibiotics per plate Mix gently For each construct mix 20 ul 10 ug of the Ready to use Packaging plasmid mix see section B for formulation with 2 ug of the plasmid lentiviral constru
14. A 1 LL AN TARAN HP skr Aer j d i EW AM LX Gef De 4 Lg E Discovery is yours CELLECTA Packaging Titering and Transduction of Lentiviral Constructs User Manual V9 5 10 2015 www cellecta com Lentiviral Packaging Titering and Transduction www cellecta com User Manual Contents A Backero nNd egene ee eee 3 B Required MEN EE 3 C Recommended Pilot Experiments ier cacecssesoed eender oe inae ae aeaii aaaea oa aaee a aiee ia aS 4 C21 DOUBLING TIME eraa e ee Meus a e a aa Ee a eea ae eee 4 C 2 Calculating a Kill ei D 5 C 3 Check Toxicity of Pohvbrene 5 CA Promoter Validation iccserecseiecee citesseceeattecanistvuceueanstessen te eraann EEE EEE Ea aE E a Eaa SE 5 D Packaging Protocol for Individual Lentiviral Constructs c ccccccccssssssssceeeeecesesseeseceeecessessaeaeeeeeens 5 D 1 Day 0 Plate Cells rcp anaa e ds aE E E E EA EE EAE aA EESE 6 D 2 Day 1 Transfection ten 10 cm platesl 7 D 3 Day 2 DNASe Treatment ccccacescccdsraccccascsecccdsanncdaatenacedcasacdeabesececesaancdcatetuce EAEEEdE KAES AE EES 7 DA Day 3 Collect Lentiviral Supernatant cccccesessssecccecessesenseeeeececsesesaaaececeessessesaeaeeeeeesssesesteaeeeeeess 8 E Concentrating Virus Optional isisccsccievecheds ssecdsevoucedadeiuecsavcoutcidevsvcadeedovcedsdedvcctavdeusessdvsveadsvveveedadssvectaveevee 8 F Transduction Lentiviral Titer Estimation cccceeseeeeeeeeeseeeeaceeeeeeeceaeeesaaeeeeaaeseeees
15. L WARRANTIES EXPRESS OR IMPLIED ORAL OR WRITTEN ARE HEREBY EXPRESSLY DISCLAIMED INCLUDING WITHOUT LIMITATION ALL IMPLIED WARRANTIES OF NON INFRINGEMENT QUIET ENJOYMENT MERCHANTABILITY OR FITNESS FOR ANY PARTICULAR PURPOSE AND ALL WARRANTIES ARISING FROM ANY COURSE OF DEALING COURSE OF PERFORMANCE OR USAGE OF TRADE Other Uses Except for the limited use expressly specified above no other license is granted no other use is permitted and CSHL retains all rights title and interests in and to the shRNA IP Rights Nothing herein confers to Customer by implication estoppel or otherwise any right or license under any patent patent application or other proprietary intellectual property right of CSHL other than the shRNA IP Rights For information on purchasing a license to use the Product for longer time periods in greater quantities or for other purposes or to practice more broadly under the shRNA IP Rights or to practice under other CSHL intellectual property rights please contact the CSHL Office of Technology Transfer at 516 367 8301 Definitions Affiliate means at the time of reference thereto any corporation company partnership joint venture or other entity which controls is controlled by or is under common control with the subject entity where control means direct or indirect ownership of more than 50 of i the outstanding stock or other voting rights entitled to elect directors or ii all ownership interests or in
16. OI 0 7 50 RFP cells If the same cells would be infected at the recommended MOI of 0 5 40 RFP cells less than 24 of RFP cells would also be puromycin resistant cells Conversely if cells were infected at MOI 2 85 RFP cells a much higher than 24 of RFP cells would also be puromycin resistant due to high of RFP cells bearing multiple integrants RFP PuroR and therefore expressing high levels of the puromycin resistance gene kd 27K NO M1 293 pure 30 fes In the case described above a 27K library genetic screen was started with at least 46 x 106 cells per replicate and transduction Cells were infected at MOI 0 7 50 transduction efficiency to obtain 23 x 10 infected RFP cells of which about 5 5 x 106 will be puro resistant 200 puro resistant cells shRNA In your screening experiment however we do not recommend Red 2nd latery A using an MOI greater than 0 5 IMPORTANT Using higher MOIs to achieve gt 40 RFP cells in order to obtain 20 or more puro resistant cells is not recommended It is advised to limit the RFP based MOI to 0 5 40 RFP cells and use enough cells at transduction to obtain the desired amount of puromycin resistant transduced cells at least 200 cells shRNA G Transduction of Target Cells Experimental Purposes G 1 General Considerations e The transduction efficiency of the lentiviral expression construct varies significantly for different cells and experimental conditions
17. PES pH7 4 and continue incubation in the CO2 incubator at 37 C for 24 hours 9 Proceed to concentration step or aliquot and store the non concentrated supernatant at 80 C Freezing and thawing usually results in 20 loss of lentiviral titer with each cycle Cellecta offers lentiviral packaging services Please contact us at sales cellecta com or visit our website at http www cellecta com for more information E Concentrating Virus Optional Although concentrating virus is optional it is recommended if 1 very high titer virus stock is needed to achieve desired MOI in hard to transduce target cells 2 virus should be suspended in another media besides DMEM 10 FBS which is optimal for sensitive target cells or 3 18h post tranduction baseline control is used in your screen to minimize problems with possible plasmid library carry over However because of the additional manipulation of samples there is the added risk of contamination and loss of virus The following protocol was optimized to concentrate virus with high recovery The protocol assumes that lentiviral supernatant was harvested 48 hours after transfection and filtered as in step 8 above 1 Aliquot lentiviral supernatant in clear sterile centrifuge tubes 2 Add LentiFuge according to the LentiFuge user manual instructions 3 Centrifuge at 15 000 x g for at least 1 hour at 4 C Mark the tubes to identify the location where the pellet will be At the
18. any country where the local law shall not permit foreign equity participation of 50 or more then the direct or indirect ownership or control of the maximum percentage of such outstanding stock voting rights or ownership interests permitted by local law Commercial Entity means any entity or organization other than a Non Profit Entity CSHL means Cold Spring Harbor Laboratory Customer means the company or other entity or organization that orders pays for and takes delivery of the Product Non Profit Entity means any college university or governmental entity including without limitation governmental and quasi governmental institutes and research laboratories or any non profit scientific research or educational organization that is of the type described in section 501 c 3 of the Internal Revenue Code or that is qualified under a state non profit organization statute Product means a product including without limitation expression vectors encoding an shRNA the design manufacture or use of which in whole or in part is the subject of the shRNA IP Rights and is deemed to include all components progeny reproductions modified versions and other derivatives thereof Seller means Cellecta Inc 2014 Cellecta Inc All Rights Reserved Trademarks CELLECTA is a registered trademark of Cellecta Inc CRL 11268 is a trademark of ATCC Invitrogen Lipofectamine and Plus Reagent are trademarks of Life Technolo
19. ate Pooled shRNA Library User Manual according to your needs available on the Cellecta website at http www cellecta com resources protocols B Required Materials e Lentiviral expression construct Custom or premade from Cellecta or generated by customer cDNA shRNA CRISPR e Lentiviral Packaging Plasmid mix Cellecta Cat CPCP K2A NOTE Cellecta s Packaging Mix is purified by double CsCl gradient In our experience CsCl purified plasmid DNA provides higher and more consistent packaging efficiencies than plasmid DNA purified by anion exchange column or other method e 293T 17 Cell Line ATCC Cat CRL 11268 e Dulbecco s Modified Eagle Medium D MEM 1X Mediatech CellGro Cat 15 013 CV NOTE ADD FRESH GLUTAMINE 1X at the time a sealed bottle of D MEM is opened even if the label indicates glutamine has already been added Glutamine in solution at 4 C has a half life of 1 2 months so glutamine D MEM purchased off the shelf from a supplier is to be regarded as glutamine In our experience the addition of glutamine increases titer approximately 2 fold If D tech cellecta com 3 of 21 v9 5 10 2015 Lentiviral Packaging Titering and Transduction www cellecta com User Manual MEM comes supplemented with stable L Alanyl L Glutamine dipeptide addition of fresh glutamine is not necessary e Glutamine L Alanyl L Glutamine Dipeptide L glutamine Mediatech Cat 25 015 CI e HEPES e MgCl
20. ct and add the plasmid mixture to 1 ml D MEM medium without serum or antibiotics Add 20 ul of Plus Reagent mix and incubate at room temperature for 15 min one 10 cm Ten 10 cm plate plates Component 60 ul 600 ul Ready to use Packaging Plasmid Mix 0 5 ug l 6 ul 60 ul Plasmid Lentiviral Construct 0 5 pg ul 1 000 ul 10 000 ul D MEM no FBS no antibiotics 20 ul 200 ul Plus Reagent 1 086 ul 10 860 ul Total volume 11X Master per plate Mix Component 1 000 ul 11 000 ul D MEM no FBS no antibiotics 30 ul 330 ul Lipofectamine 1 030 ul 11 330 ul Total volume We recommend making enough master mix for one extra reaction to compensate for pipette error 5 Add 1 ml of the diluted Lipofectamine Reagent from step 4 to the DNA Plus Reagent complex from step 3 mix gently by flicking the tube or vortexing and incubate at room temperature for 15 min 6 Add 2 ml of the DNA Plus Reagent Lipofectamine Reagent complex from step 5 to each 10 cm plate from step 2 and mix complexes with medium by gentle rotation Take care not to dislodge cells from the plate Incubate at 37 C in the CO2 incubator for 24 hours D 3 Day 2 DNAse I Treatment 7 tech cellecta com At 24 hours post transfection replace the medium containing complexes with fresh 10 ml D MEM medium supplemented with 10 FBS DNase I 1 U ml MgCl2 5 mM 20MM HEPES pH7 4 Continue inc
21. dia 24 hours before harvesting and make sure to supplement media with HEPES pH 7 4 20mM final H 2 Poor transduction efficiency Problem Target cells have too high or too low density Solution Plate fewer or more cells in order to have 20 50 confluency at transduction stage Problem Target cell line may be difficult to transduce Solutions 1 Use a higher concentration of lentiviral particles 2 Perform Spinoculation to improve transduction efficiency 3 Check to see if Polybrene was added at 5 pg ml Problem Wrong amount of Polybrene added during transduction stage Solution If Polybrene is toxic to the target cells optimize Polybrene concentration in the range of 0 5 ug ml by performing a toxicity titration as described in Calculating a Kill Curve Most vectors from Cellecta that are used to make pooled shRNA libraries have an antibiotic resistance gene which allows you to select the cells that have received a copy of the shRNA In order to successfully select your cells you need to know the concentration of antibiotic that kills your untransduced cells within 72 hours Antibiotic selection is not necessary for most screens but it is a convenient way of removing excess cells that have not received the lentiviral vector It is helpful to use minimal levels of antibiotic so as not to kill cells that just have a weaker expression of the antibiotic resistance gene Many vectors contain a puromycin resistance gene there
22. e creation of splashes or aerosols e Work surfaces are decontaminated at least once a day and after any spill of viable material e All cultures stocks and other regulated wastes are decontaminated before disposal by an approved decontamination method such as autoclaving Materials to be decontaminated outside of the immediate laboratory area are to be placed in a durable leakproof properly marked biohazard infectious waste container and sealed for transportation from the laboratory tech cellecta com 19 of 21 v9 5 10 2015 Lentiviral Packaging Titering and Transduction www cellecta com User Manual K References For a complete list of References and Product Citations please see http www cellecta com resources publications L Terms and Conditions Cellecta Inc Limited License Cellecta grants the end user the Recipient of the Pooled Lentiviral shRNA Libraries and Vector the Product a non transferable non exclusive license to use the reagents for internal research use only as described in the enclosed protocols in particular research use only excludes and without limitation resale repackaging or use for the making or selling of any commercial product or service without the written approval of Cellecta Inc separate licenses are available for non research use or applications The Product is not to be used for human diagnostics or included used in any drug intended for human use Care and at
23. eaeeeeaaeeeeaeeeeaes 9 F 1 Transduction for Lentiviral Tltering nserita iea aikoa aaa RE i Eae 9 F 1 1 Transduction of Adherent Cells HEK293 cells Titering ccccccccssssecesssceceesssseeeesseseeeesseaeees 9 F 1 2 Alternative Transduction Protocol Spinoculation for Cells in Suspension Titering 10 F 2 Lentiviral Titer estimation RFP assay ccccccccssccccesssececeeaeceesesaeeecsesaeeeceesaeeeesesaeceesesaeceesesaeeeeneaas 10 F 3 Calculating the PUrOMYCiN Titer cccccccccesccsscsscsecssccsscsecsescsscsucseesssseusesceususcsessesseusensaeseusensaseas 12 G Transduction of Target Cells Experimental Purposes 13 G 1 General Consl eratlopg cu ERENNERT eeh geeiert ege d ege nates 13 G 2 Transduction Protocol HEK293 Easy to Transduce Gelle 14 EEN Day DV EE 14 G22 DAY 2 EE 14 G 3 Tet Regulated COMStCUCtS ccsceccccces cisecgcacececss sohsctadeceeesesscbaccacacceeassavdacaacecsendssawbaecacncsesadee ectadecessasedede 15 G 3 1 Day 5 tet regulated constructsl cccesccceessscececsseceseeseeeceesaececeesseeeceesaeeeseesaeeeseesaeeeeeesaeeeeees 15 G 3 2 Day 7 tet regulated constructs cccesccceesssceceessececeeseeeceesseeeceesseeecsesaeeeceesaeeeceeaeeeeeeseeeeeees 15 G 4 Non Tet Regulated Constructs ccccccccccscssssssssecececessesesneaeeeeeesseesesaeeeeeeeceseeseeaeeeseesessseaeaeeeesesseseees 15 G 4 1 Day 5 NON tet regulated constructsl 15 G 4 2 Day 6 or 7 NON tet regulated cons
24. erred to is Xe The cells should be in the log phase of growth to calculate doubling time properly so it is important to not let the cells become confluent To calculate the doubling time T In2 Xe In GD Doubling Time where T Time in any units in this case days For Example let s say that on Day 0 you count 2x10 cells Three 3 days later you count the cells at 16x106 cells Xb 2x106 T 3 days Xe 16x106 tech cellecta com 4 of 21 v9 5 10 2015 Lentiviral Packaging Titering and Transduction www cellecta com User Manual poublina Time Mini _ 3 0 69 _ 2 08 _ OU ing me 16 000 000 wen 2 08 ay n gt 00 0002 C 2 Calculating a Kill Curve Most vectors from Cellecta that are used to make pooled shRNA libraries have an antibiotic resistance gene which allows you to select the cells that have received a copy of the shRNA In order to successfully select your cells you need to know the concentration of antibiotic that kills your untransduced cells within 72 hours Antibiotic selection is not necessary for most screens but it is a convenient way of removing excess cells that have not received the lentiviral vector It is helpful to use minimal levels of antibiotic so as not to kill cells that just have a weaker expression of the antibiotic resistance gene Many vectors contain a puromycin resistance gene therefore we will use this example as to the method of calculating a puromycin kill curve Aliquo
25. fore we will use this example as to the method of calculating a puromycin kill curve Aliquot cells in a 12 well plate at such a density so they are at 72 hours from confluency use the doubling time calculation to help you determine the amount of time this will take Add puromycin at 0 ug ml 0 5 pg ml 1 ug ml 2 ug ml 5 ug ml and 10 ug ml in six different wells Mix and return cells to incubator Grow cells under standard conditions for 48 72 hours For puromycin use the lowest concentration that kills gt 90 of cells in 48 72 hours C 3 Check Toxicity of Polybrene Section Problem Loss of lentiviral titer during storage Solution Ensure storage of aliquoted packaged shRNA library at 80 C Each freeze thaw cycle typically causes reduction of the titer by 20 Use a fresh stock for transduction Problem The RFP assay is performed too early tech cellecta com 16 of 21 v9 5 10 2015 Lentiviral Packaging Titering and Transduction www cellecta com User Manual Solution Normally the maximal expression of RFP from the integrated provirus is expected to develop by 72 hours after transduction However some cells exhibit delayed expression Try the assay at a later time such as 96 hours Problem The RFP assay is performed with the wrong flow cytometry settings Solution RFP cells are to be detected using a 56inm laser for excitation 530nm still acceptable and 600 20 band pass filters or similar for detection for TagRFP
26. gies Corporation tech cellecta com 21 of 21 v9 5 10 2015
27. l additional complete medium per well no Polybrene 5 24 hours after spinoculation resuspend cells at 2 x 10 cells ml in RPMI 10 FBS in the appropriate culture vessel and grow for additional 48 hours 6 72 hours after spinoculation perform titer as previously described NOTE Use larger vessels for large scale genetic screen transductions Scale up all volumes accordingly F 2 Lentiviral Titer estimation RFP assay Lentiviral shRNA vectors that express the fluorescent protein TagRFP excitation 560nm emission 590nm allow lentiviral titer estimation by flow cytometry RFP assay or by a combined flow cytometry puromycin resistance assay RFP Puro assay To check lentiviral titer we recommend always using the same cells you will use in the screen Most of the commonly used mammalian cell lines can be effectively transduced by lentiviral constructs Relative titers can vary up to 50 fold depending on the chosen cell line Lentiviral titer is measured as Transduction Units ml TU ml One TU produces one integration event in target cells Integration events can be calculated from observed of transduced cells according to the table below tech cellecta com 10 of 21 v9 5 10 2015 Lentiviral Packaging Titering and Transduction www cellecta com User Manual TITER CHART 100 5 90 80 70 60 50 40 infected cells 30 20 10
28. ng on the growth area of the well or plate and the growth characteristics of the cell type G 2 1 Day 1 1 Quickly thaw the lentiviral vector particles in a water bath at 37 C Transfer the thawed particles to a laminar flow hood gently mix by rotation inversion or gentle vortexing and keep on ice CAUTION Only open the tube containing the lentiviral vector particles in the laminar flow hood NOTE Unused lentiviral stock may be refrozen at 80 C but it will typically result in a loss of about 20 in titer 2 Trypsinize and resuspend cells to a density of 1 x 10 cells ml in D MEM supplemented with 10 FBS and 5 ug ml Polybrene Aliquot 1 ml well in a 12 well plate and add an appropriate amount of lentiviral vector stock to each well For one well mock well control do not add lentiviral vectors Mix and return cells to CO2 incubator Grow cells under standard conditions for 24 hours Note For shRNA constructs we recommend starting with an MOI of about 1 ratio of lentiviral vectors to cells In some cases a higher MOI or multiple integrants per cell may be needed to reach an optimal knockdown efficiency For CRISPR constructs which are large you may need to increase the MOI to achieve efficient transduction efficiency G 2 2 Day 2 At 24 hours post transduction replace media with fresh D MEM supplemented with 10 FBS and without Polybrene Return cells to CO2 incubator and grow under standard conditions for 24 more hour
29. nt provided by the Third Parties Agilent Technologies Inc End User Label License for the use of shRNA libraries comprising Oligo Pools This Internal Use only license grants End Users the sole right to use and fully consume or destroy this product the Product Use of the Product is limited to Research Use ONLY not for diagnostic procedures In all cases sale or other transfer or distribution to third parties of i the Product or any portion ii DNA RNA and protein constructs or libraries created from the Product or any portion or of iii transformed phage viruses cells or tissues created directly or indirectly from the Product or any portion is strictly prohibited without prior written approval by Agilent Technologies Inc Life Technologies Corporation End User Label License for the use of Lentiviral Expression System This product or service based upon the Lentiviral Expression System is sublicensed from Life Technologies Corporation under U S Patent Nos 5 686 279 5 834 256 5 858 740 5 994 136 6 013 516 6 051 427 6 165 782 6 218 187 6 428 953 6 924 144 7 083 981 and 7 250 299 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 nonhuman research use requires a license from GBP IP LLC Please contact GBP IP LLC 537 Steamboat Road Suite 200 Greenwich CT 06830 Use of thi
30. ol Spinoculation for Cells in Suspension Titering The following protocol has been optimized for K 562 cells For other cell types parameters such as media growth surface time of detection etc will have to be adjusted 1 K 562 cells are transduced infected using spinoculation This is performed using multi well tissue culture plates and a tabletop centrifuge capable of 1 200 x g and centrifugation of multi well plates 2 Grow K 562 cells and maintain them between 2 x 10 and 1 x 106 cells ml Do not let them become too dense or let the medium become yellow at any point 3 For lentiviral library titration K 562 cells are resuspended at 2 x 106 cells per ml in RPMI 10 FBS supplemented with 20mM HEPES pH7 4 and Polybrene 5 ug ml 0 5 ml aliquots are placed into each well in a 24 well plate 1 x 10 cells well total This cell density has proven effective for many suspension cell lines in house at Cellecta To each cell containing well add increasing amounts of lentiviral stock to be titered For standard 100 fold concentrated lentiviral stock add 0 ul 0 3 ul 1 ul 3 ul and 10 ul virus Close the plate mix by gentle agitation wrap the perimeter with parafilm and place the plate into centrifuge with an appropriate balance and centrifuge at 1 200 x g at 25 C for 2 hours 4 Following centrifugation remove plate s from centrifuge carefully remove parafilm and place in incubator After 3 hours feed cells with 0 5 m
31. ow many replicates did you use i e duplicate triplicate etc Did you use puromycin after transduction and at what concentration For how long did you use puromycin on the cells Please refer to the questions above and contact us by phone or email Phone 1 650 938 3910 Toll Free 1 877 938 3910 Fax 1 650 938 3911 E mail Technical Support tech cellecta com General Information info cellecta com Sales sales cellecta com Orders orders cellecta com Blog http www cellecta com blog Postal Mail Cellecta Inc 320 Logue Ave Mountain View CA 94043 For more information about Cellecta s products and services please visit our web site at http www cellecta com tech cellecta com 18 of 21 v9 5 10 2015 Lentiviral Packaging Titering and Transduction www cellecta com User Manual J Safety Guidelines The HIV based lentivector system is designed to maximize its biosafety features which include e A deletion in the enhancer of the U3 region of 3 ALTR ensures self inactivation of the lentiviral construct after transduction and integration into genomic DNA of the target cells e The RSV promoter upstream of 5 LTR in the lentivector allows efficient Tat independent production of lentiviral RNA reducing the number of genes from HIV 1 that are used in this system e Number of lentiviral genes necessary for packaging replication and transduction is reduced to three gag pol
32. r Manual Once titer is estimated the amount of Lentiviral Stock necessary to transduce any given of target cells at any transduction efficiency range of 10 80 infected cells can be back calculated from the TITER FORMULA and TITER CHART above Example To transduce 20 000 000 cells at 50 transduction efficiency with a Lentiviral Stock titer of 3 000 000 TU ml we calculated the required amount of Lentiviral Stock as follows We calculate the required MOI to achieve 50 transduction efficiency using the TITER CHART 50 transduction efficiency 0 7 MOI We calculate the volume of Lentiviral Stock required using the TITER FORMULA f ll t Tr ducti X L 7 oj celisa ansauction I f Viral Stock use tT S cti ml 0 7 ml of Viral Stock used at Transduction 3 000 000 20 000 000 Viral Stock 20 000 000 4 67 ml 3 000 000 F 3 Calculating the Puromycin Titer If puromycin selection of transduced cells is going to be performed in the screen the fraction of RFP cells at a given MOI that will survive puromycin selection must be calculated beforehand Even though RFP and Puro resistance markers are expressed from the same promoter not all cells expressing detectable RFP are guaranteed to be puro resistant A threshold of Puro expression is required to confer puromycin resistance Depending on cell type such a threshold is associated with different levels of RFP co expression Depending on the MOI used a
33. rev The corresponding proteins are expressed from different plasmids lacking packaging signals and share no significant homology to any of the expression lentivectors pVSV G expression vector or any other vector to prevent generation of recombinant replication competent virus e None of the HIV 1 genes gag pol rev are present in the packaged lentiviral genome as they are expressed from packaging plasmids lacking packaging signal therefore the lentiviral particles generated are replication incompetent e Lentiviral particles will carry only a copy of your expression construct Despite the above safety features use of HIV based vectors falls within NIH Biosafety Level 2 criteria due to the potential biohazard risk of possible recombination with endogenous lentiviral sequences to form self replicating virus or the possibility of insertional mutagenesis For a description of laboratory biosafety level criteria consult the Centers for Disease Control Office of Health and Safety Web site at http www cdc gov biosafety publications bmbI5 bmbI5_sect_iv pdf It is also important to check with the health and safety guidelines at your institution regarding the use of lentiviruses and follow standard microbiological practices which include e Wear gloves and lab coat at all times when conducting the procedure e Always work with lentiviral particles in a Class II laminar flow hood e All procedures are performed carefully to minimize th
34. s G 2 3 Day 3 By day 3 the culture may be confluent depending on cell type The cells can be assayed for RFP or GFP expression at this point Split the cells 1 4 to 1 8 depending on the type of cells and incubate for 48 hours in complete medium containing antibiotic puromycin bleomycin hygromycin etc at 1 ug ml or the concentration determined empirically by the Antibiotic Kill Curve tech cellecta com 14 of 21 v9 5 10 2015 Lentiviral Packaging Titering and Transduction www cellecta com User Manual G 3 Tet Regulated Constructs G 3 1 Day 5 tet regulated constructs Change media to remove dead cells dilute culture as needed to keep cells from confluency split culture into 2 separate samples and incubate 48 hours in complete medium with Puromycin with one sample or without the other sample 0 1 ug ml dox G 3 2 Day 7 tet regulated constructs The infected target cells can now be analyzed using an appropriate assay If a prolonged shRNA expression is required it is recommend that cells are kept under optimal growth conditions and fed fresh media containing 0 1 ug ml dox every 48 hours until harvesting G 4 Non Tet Regulated Constructs G 4 1 Day 5 NON tet regulated constructs Change media to remove dead cells dilute culture as needed to keep cells from confluence and incubate 24 48 hours in complete medium with Puromycin G 4 2 Day 6 or 7 NON tet regulated constructs The infected target cells
35. s technology to make or sell products or offer services for consideration in the research market requires a license from Life Technologies Corporation 5791 Van Allen Way Carlsbad CA 92008 Evrogen IP JSC End User Label License for the use of lentiviral shRNA constructs comprising TagRFP encoded gene This product is for internal non commercial research use only No rights are conveyed to modify or clone the gene encoding fluorescent protein contained in this product The right to use this product specifically excludes the right to validate or screen compounds For information on commercial licensing contact Evrogen Licensing Department email license evrogen com Cold Spring Harbor Laboratory CSHL End User Label License for use of expression vectors encoding an shRNA Acceptance This Limited Use License License contains the exclusive terms and conditions between CSHL and Customer for use of the Product By opening the Product container or in any other way accessing or using the Product Acceptance you will create a binding legal contract upon the terms and conditions herein without modification Customer s purchase order or similar terms shall not apply to this License If you are not authorized by Customer to enter into this License or do not agree to all terms and conditions in this License then you are prohibited from opening the Product container or otherwise accessing or using the Product Permitted Use
36. t cells in a 12 well plate at such a density so they are at 72 hours from confluency use the doubling time calculation to help you determine the amount of time this will take Add puromycin at 0 ug ml 0 5 ug ml 1 ug ml 2 ug ml 5 ug ml and 10 ug ml in six different wells Mix and return cells to incubator Grow cells under standard conditions for 48 72 hours For puromycin use the lowest concentration that kills gt 90 of cells in 48 72 hours C 3 Check Toxicity of Polybrene Polybrene is a transduction enhancement reagent used during transduction of lentiviral particles into the target cells Polybrene is a polycation that neutralizes charge interactions to increase binding between the lentiviral envelope and the plasma membrane The optimal concentration of Polybrene depends on cell type and may need to be empirically determined Excessive exposure to Polybrene can be toxic to some cells Before transducing your target cells we recommended performing a Polybrene toxicity titration In a 12 well plate grow cells in complete culture medium with a range of Polybrene concentrations 0 pg ml 1 ug ml 2 ug ml 3 ug ml 4 ug ml 5 ug ml for 24 hours Then replace old medium with Polybrene free complete culture medium and grow cells for an additional 72 hours Check for toxicity by counting viable cells For your experiments use the highest concentration of Polybrene that results in less than 10 cell toxicity compared to no Polybrene typically
37. tention should be exercised in handling the Product by following appropriate research laboratory practices Cellecta s liability is expressly limited to replacement of Product or a refund limited to the actual purchase price Cellecta s liability does not extend to any damages arising from use or improper use of the Product or losses associated with the use of additional materials or reagents This limited warranty is the sole and exclusive warranty Cellecta does not provide any other warranties of any kind expressed or implied including the merchantability or fitness of the Product for a particular purpose Use of the Product for any use other than described expressly herein may be covered by patents or subject to rights other than those mentioned Cellecta disclaims any and all responsibility for injury or damage that may be caused by the failure of the Recipient or any other person to use the Product in accordance with the terms and conditions outlined herein The Recipient may refuse these licenses by returning the enclosed Product unused By keeping or using the enclosed Product you agree to be bound by the terms of these licenses The laws of the State of California shall govern the interpretation and enforcement of the terms of these Licenses Limited Use Label Licenses The Recipient acknowledges that the Product has been developed by Cellecta based on licenses from Third Parties and agrees with the Terms of Limited Use for the Recipie
38. to accurately record the original of cells at Time of Transduction as this is critical in titer calculation For adherent cells other than HEK293 choose a different of cells at time of transduction depending on cell size As a rule of thumb cells should be transduced at such a density such that they would become confluent in 48 hours For example for HeLa cells the suggested cell is 50 000 cells well in a 12 well plate Day 2 3 Between 16h 24 hours post transduction replace media with fresh D MEM supplemented with 10 FBS and without Polybrene Return cells to CO2 incubator and grow under standard conditions for additional 48 hours Avoid confluence trypsinize and re plate cells if needed Day 4 72 hours after transduction 4 Detach cells from the plate by trypsin treatment block trypsin with FBS media centrifuge resuspend in 1X D PBS and determine the of transduced RFP positive cells by flow cytometry NOTE Attempting to determine the of transduced cells by fluorescence microscopy is NOT RECOMMENDED tech cellecta com 9 of 21 v9 5 10 2015 Lentiviral Packaging Titering and Transduction www cellecta com User Manual IMPORTANT Flow cytometry settings to detect RFP positive cells are the following Excitation 561inm 530nm laser is still acceptable Emission 600 20 band pass filter or similar for TagRFP 5 Proceed to Lentiviral Titer estimation RFP assay F 1 2 Alternative Transduction Protoc
39. tructs cccccsccceessececeesseceeeeseeeeceeaeeeceesseeeceeaeeeeeesaeeeeeees 15 tech cellecta com 2 of 21 v9 5 10 2015 Lentiviral Packaging Titering and Transduction www cellecta com User Manual H AROE oLa e E E E E ERE E E E AE A E E E ET 15 H 1 Low Lentiviral Titer lt 10 TU ml in supernatant 15 Hi1 1 Poor transfection GhFICIENCY eege eege Nee Eege Nee 15 H 1 2 Inefficient production Of the virus 15 H 2 POOr transduction Mee 16 H 3 Transduction affects target cell Viability ccccccccccscsssesssceeececeeseseeseseeeeeesssesesaeeeeeeseesseseeaees 17 HA No expression of RFP or Puro or ShRNAs in target cells ccccccscecsscescssecsssesscsessesessssesseeeeess 17 l TECHNIGal SUP POM EE 18 J Sate GUIdEliNE ee EE Ee 19 K References sek eae iecibidns a EE E ese eee eee 20 L Terms and eut e EE 20 A Background The protocols below provide the instructions on how to package titer and transduce target cells with lentiviral expression constructs The protocols here are useful for any type of lentiviral construct including cDNA expression constructs shRNA expression constructs CRISPR constructs or reporter vectors Please read the entire user manual before proceeding with your experiment To ensure you have the latest version of this user manual please check the Cellecta website at http www cellecta com resources protocols For packaging of Cellecta Pooled shRNA Libraries please see the appropri
40. ubation in the CO2 incubator at 37 C overnight Overnight DNase I treatment before harvesting virus does not negatively affect lentiviral titer or infectivity and helps prevent undesirable carryover of plasmid construct into the virus prep 7 of 21 v9 5 10 2015 Lentiviral Packaging Titering and Transduction www cellecta com User Manual NOTE Failure to change the media the day after transfection results in large carryover of plasmid free and or Lipofectamine bound in your lentiviral prep This may cause problems with most downstream molecular biology applications especially whenever there is a PCR step involved DA Day 3 Collect Lentiviral Supernatant A CAUTION You are working with infectious lentiviral particles at this stage Please follow the recommended guidelines for working with BSL 2 safety class materials see Safety Guidelines 8 At 48 hours post transfection collect all 10 ml of the virus containing medium from each plate and filter the supernatant through a Nalgene 0 2 um PES filter a low protein binding filter to remove debris and floating packaging cells Failure to filter supernatant could result in carry over of cells into your lentiviral prep NOTE Usually the peak of virus production is achieved at 48 hours post transfection Supernatant can also be collected again at 72 hours post transfection replace the collected 48 hour supernatant with 10 ml of fresh D MEM medium supplemented with 10 FBS 20mM HE
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