Enhanced Episome Vectors (EEV)
Contents
1. The EEV605A 1 cumate switch inducible reporter Page 6 ver 1 012915 www systembio com EEV Episomal Vector System Cat s EEV6xxA 1 should be silent until the addition of the inducer cumate solution Cat QM150A 1 1000x concentration Cumate was added at 1x concentration from the 1000x stock daily at a final concentration in the medium of 30mg ml and cells imaged for GFP induction The GFP induction appeared after 2 days and then expression was detected for 72 days 2 5 months after the original transfection under 2 ug ml puromycin selection The GFP induction data for the no cumate and the 11 and 72 day expression data are shown in the next Figure Cuo GFP T2A Luc EEV605A 1 Reporter No Cumate Cumate 1 1 Days Cumate 72 Days 888 266 5066 Toll Free 650 968 2200 outside US Page 7 System Biosciences SBI User Manual The expression activity of the EEV605A 1 reporter plasmid upon cumate induction can also be monitored in living cells for luciferase expression Simply add D luciferin to the cell media for 2 3 minutes and image the cells for luminescence An example is shown below of a 6 well dish of HEK298T cells transfected with EEV605A 1 as well as control untransfected cells Both conditions then were either treated with 1x cumate solution or no cumate Cells were subcultured in cumate for 27 days and imaged for 120 seconds to detect luminescence using a Bio Rad Chemi doc system The resulting cumate induct2. and GS enhances transient protein expression Biotechnol Prog 2014 Jan Feb 30 1 132 41 John L Yates Noreen Warren amp Bill Sugden Stable replication of plasmids derived from Epstein Barr virus in various mammalian cells Nature 313 812 815 28 February 1985 doi 10 1038 313812a0 Mark A Kay Cheng Yi He amp Zhi Ying Chen A robust system for production of minicircle DNA vectors Nat Biotechnol 2010 Dec 28 12 1287 9 Lu J Zhang F Kay MA A mini intronic plasmid MIP a novel robust transgene expression vector in vivo and in vitro Mol Ther 2013 May 21 5 954 63 V Technical Support For more information about SBI products and to download manuals in PDF format please visit our web site http Awww systembio com For additional information or technical assistance please call or email us at System Biosciences SBI 265 North Whisman Rd Mountain View CA 94043 Phone 650 968 2200 SBI 888 266 5066 Toll Free Fax 650 968 2277 E mails General Information info systembio com Page 14 ver 1 012915 www systembio com Vil EEV Episomal Vector System Cat s EEV6xxA 1 Technical Support tech systembio com Ordering Information orders systembio com Licensing and Warranty information Please note the following Licensing Restrictions For Academic and Non Profit Institutions Researchers at academic and non profit institutes are granted full access to purchasing the EEV cloning vectors
3. and reporters Full sequence information is provided upon proof of purchase of EEV vectors Please contact SBI s_ technical support at tech systembio com for sequence information For Commercial Customers For profit and commercial customers can purchase the pre made EEV reporters EEV604A 1 EEV605A 1 however due to licensing restrictions cloning EEV vectors e g EEV600A 1 and EEV610A 1 are not available for purchase unless a custom EEV construct is purchased as part of a custom service SBI then provides the commercial customer with the desired amount of ready to use EEV custom construct DNA Limited Use License Use of the EEV vector system i e the Product is subject to the following terms and conditions If the terms and conditions are not acceptable return all components of the Product to System Biosciences SBI within 7 calendar days Purchase and use of any part of the Product constitutes acceptance of the above terms The purchaser of the Product is granted a limited license to use the Product under the following terms and conditions 888 266 5066 Toll Free 650 968 2200 outside US Page 15 System Biosciences SBI User Manual e The Product shall be used by the purchaser for internal research purposes only The Product is expressly not designed intended or warranted for use in humans or for therapeutic or diagnostic use e The Product may not be resold modified for resale or used to manufacture com
4. 3 SBI System Biosciences Enhanced Episome Vectors EEV Cat s EEVxxxA 1 User Manual Store at 20 C upon arrival A limited use label license covers this product By use of this product you accept the terms and conditions outlined in the Licensing and Warranty Statement contained in this user manual EEV Episomal Vector System Cat s EEV6xxA 1 Contents Is SIMTRODUCHON eataa e aa e 1 A Enhanced Episomal Vectors EEV Overview 0 2 B EEV Applications and Vector Formats 3 Il EEV Reporters and Sample Data 0 cccccessceseeeeeeseeeeeneeees 5 Constitutive and Inducible EEV Reporter Construct Maps 5 B Sample EEV Reporter Expression Data cc cccceeseees 6 III Product Information and Protocols 12 IV Frequently Asked Questions cccesceeeeeeeseeeeeeeeeeenees 13 V References wea titi enti ia ai eee ae 14 Vie Technical Supporti a a a aaa aaa ret 14 VII Licensing and Warranty information ccccceeeeeeees 15 888 266 5066 Toll Free 650 968 2200 outside US Page 1 System Biosciences SBI User Manual A Enhanced Episomal Vectors EEV Overview For many years expression vectors have been used as tools for providing transgenic information into mammalian cultured cells and therapeutic studies For example reprogramming factors were shown to be delivered through retroviral transduction in cells allowing any somatic cell to differentiate much like hu
5. DD injection with subsequent monitoring of secreted mIL 23 protein levels in the serum of test mice Two different amounts of either minicircle mini intronic platform or EEV plasmid DNA expressing the mIL 23 gene were utilized for these cross comparison studies Serum levels of mIL 23 expression were quantitated using a murine IL 23 ELISA assay 7 days after HDD injection of the various plasmid DNAs 888 266 5066 Toll Free 650 968 2200 outside US Page 9 System Biosciences SBI User Manual AmpR CAGs Promoter ColE1 ori CAGs mIL 23 in EEV600A 1 11846 bp WPRE MC Minicircle DNA polyA MIP Mini Intronic Plasmid DNA EEV Enhanced Episome DNA EBNA1 1 2x10 6 3 Day 7 IL 23 serum levels 800000 400000 120000 60000 30000 0 Serum IL 23 levels pg mL oy SF X SP C EE S SLES The EEV construct map of the mIL 23 EEV expression construct cloned into EEV600A 1 and the mIL 23 serum expression data are shown above The EEV technology outperformed the other episomal platforms by at least 10 fold over the course of these studies up to 21 days post HDD injection not shown This demonstrates the ability of the EEV platform to provide sustained long lasting transgene expression which is critical for endpoint studies requiring weeks or even months of transgene expression The Cumate inducible EEV605A 1 reporter was also tested in mice The EEV605A 1 CuO GFP T2A Luciferase plasmid was introduced into te
6. V reporter plasmids featuring a GFP T2A Luciferase expression cassette for reporting sustained transgene expression in a constitutive format cat EEV604A 1 or as an all in one cumate inducible format cat EEV605A 1 The construct plasmid maps are depicted below Constitutive Reporter AmpR CAGs Promoter ColE1 ori CAGs GFP Luc cat EEV604A 1 were 12792 bp EBNA1 Inducible Reporter AmpR CuO Promoter ColE1 ori GFP Luciferase CuO GFP Luc cat EEV605A 1 Y WPRE 16680 bp EF1 promoter CymR EBNA1 polyA 888 266 5066 Toll Free 650 968 2200 outside US Page 5 System Biosciences SBI User Manual B Sample EEV Reporter Expression Data EEV in vitro applications The constitutive EEV reporter construct cat EEV604A 1 was transfected into HEK293T cells and the GFP transgene expression monitored over a period of several weeks HEK293T cells in a 24 well culture dish were transfected once with 0 5 ug of EEV604A 1 reporter plasmid DNA Cell images from 2 days and 11 days after transfections are shown below EEV604A 1 CAGs OP NAA LURKERS The cumate inducible EEV reporter construct cat EEV605A 1 features a cumate inducible promoter driving the expression of GFP T2A Luciferase as well as a constitutive EF1 promoter expressing the CymR cumate promoter repressor and a Puromycin selection cassette HEK293 cells were transfected with EEV605A 1 Cells were cultured with DMEM supplemented with 10 FBS
7. al Vector System Cat s EEV6xxA 1 e EEV610A 1 EEV605A 1 are inducible systems that have a cumate promoter and a puromycin marker for selecting and establishing cell lines e EEV604A 1 and EEV605A 1 also feature a GFP T2A Luciferase reporter to monitor expression of EEV both in vitro and in vivo animal studies lll Frequently Asked Questions Q What is the cDNA maximum size that can be expressed from an EEV plasmid Although the insert size limits have not been thoroughly tested we have had success in expressing up to a 12 kb insert at high levels using the EEV system Q Will the EEV plasmids work in Human Mouse and Rat cells Yes the EBNA1 and oriP vector technologies will work in most mammalian cells and tissue types Q How can I control reduce the expression level of my cloned cDNA in an EEV plasmid If your cDNA is cloned into the constitutive expression plasmid cat EEV600A 1 then simply titrate down the amount of EEV construct DNA you use for cell transfections and or animal injection studies Alternatively you can use the cumate inducible EEV format cat EEV610A 1 and titrate in the amount of cumate added to achieve the desired expression level 888 266 5066 Toll Free 650 968 2200 outside US Page 13 System Biosciences SBI User Manual IV References Daramola O Stevenson J Dean G Hatton D Pettman G Holmes W Field R A high yielding CHO transient system coexpression of genes encoding EBNA 1
8. arranty SBI does not provide any other warranties of any kind expressed or implied including the merchantability or fitness of the Product for a particular purpose Page 16 ver 1 012915 www systembio com EEV Episomal Vector System Cat s EEV6xxA 1 SBI is committed to providing our customers with high quality products If you should have any questions or concerns about any SBI products please contact us at 888 266 5066 2015 System Biosciences SBI All Rights Reserved 888 266 5066 Toll Free 650 968 2200 outside US Page 17
9. chnology enables sustained transgene expression for several months in both in vitro and in vivo applications The mechanism of how the EEV technology works is shown in the next schematic Page 2 ver 1 012915 www systembio com EEV Episomal Vector System Cat s EEV6xxA 1 Transfect EEV Plasmid i A A pa into cells i EEV poraa nonviral isomal non integrating Vector mums xe i agar paS A expression AI i 8 J EEV GFP up to 3 months EEV plasmids anchor to the host chromatin and replicate during the cell cycle division B EEV Applications and Vector Formats EEV vectors are a non viral and non integrating system for sustained transgene expression in cells The built in oriP EBNA1 gene in these vectors allows these plasmids to be replicated and partitioned to daughter cells As a result this EEV system is optimized to be expressed in cell culture and in vivo animal models for up to 6 weeks The EEV platform has been utilized by SBI to rapidly and efficiently reprogram somatic cells into iPSCs for additional information please see product description for SBI s Episomal iPSC Reprogramming Plasmids cat SC900A 1 The EEV system is optimal for researchers looking for a non viral non integrating system to provide long term stable transgene expression in target cells or tissues and offers clear advantages over both viral and traditional plasmid based expression systems Highlights of the EEV System e Easy to cl
10. g uL with 20 ug total All plasmids are shipped in dry ice or blue ice and should be stored at 20 C upon receipt Properly stored plasmids are stable for 12 months from the date received Building EEV expression constructs Standard molecular cloning techniques to insert the cDNA microRNA etc of interest can be used and placed into the MCS of the EEV cloning and expression vector Typical ampicillin based plates and liquid media can be used to identify successful clones and propagate the EEV plasmids Transfection of EEV vectors in Tissue Culture For 293 FT cells in a 24 well plate format e One day prior to transfection seed 1 0 x 10 5 cells well in a 24 well plate e Cells should be 60 80 confluent on day of transfection Replace cells with fresh new media at least 2 hours prior to transfection Transfection method recommended 1 Per 24 well to be transfected 2 First dilute 0 5 ug EEV plasmid in 100 uL Dilution media Serum Free then mix well 3 Add 2 3 uL of Lipofectamine LTX reagent and mix by gently flicking the tube and spin down briefly 4 Incubate at 25 C for at least 30 minutes 5 Then take 100 uL of transfection mix and add drop wise to the cells 6 Let plasmid express for 24 72 hours Establishing EEV cell lines e EEV600A 1 and EEV604A 1 are constitutive promoters which allow sustained expression of EEV plasmids and target gene for at least 6 weeks Page 12 ver 1 012915 www systembio com EEV Episom
11. ion data in living cells with the EEV605A 1 reporter are shown in the next Figure Cumate Cumate White Light Luminescence HEK293 cells 27 days cumate induction D Luciferin 150ug mL EEV in vivo applications The constitutive EEV604A 1 CAGs GFP T2A Luciferase construct 8 ug plasmid DNA was introduced into test mice through hydrodynamic tail vein injection HDD This procedure leads to high plasmid DNA transfection of the livers of mice in vivo The test mice n 3 were imaged for body luminescence in the liver area post HDD from Day 1 up until Day 80 The results show that robust EEV luciferase expression is readily detectable at very high Page 8 ver 1 012915 www systembio com EEV Episomal Vector System Cat s EEV6xxA 1 levels through Day 40 Luciferase quantitative data for the test mice in graphical form as well as bioluminescent animal imaging data are shown in the next Figure EEV604A 1 CAGs GFP T2A Luciferase m Day 20 Day 80 _ Q o Counts photons sec Day 30 Day 40 Day 60 There are some popular episomal expression technologies available for sustained expression in vivo such as the Minicircle DNA system Mark A Kay et al Nat Biotechnol 2010 Dec 28 12 1287 9 and Mini Intronic Platform Lu J et al Mol Ther 2013 May 21 5 954 63 These two different episomal technology platforms were compared against the EEV system using a mouse IL 23 cDNA followed by H
12. man ES cells However viral transduction systems have many challenges for in vivo studies due to potential for random integration and mutagenesis To avoid the challenges of viral transduction systems nonviral non integrating plasmid based expression and reprogramming vectors such as those based on the Epstein Barr Nuclear Antigen 1 oriP EBNA1 have been developed They have been validated to efficiently reprogram fibroblasts into induced Pluripotent Stem Cells iPSCs without integrating or modifying the host s genome SBI s catalog SC900A 1 The major distinguishing feature of the EBNA system from viral or other plasmid based approaches is its ability to replicate in synchrony with the host genome by attaching to the host chromatin and replicate with each cell cycle division This results in an extended presence within a host cell The target gene to be expressed such as a reporter or iPSC reprogramming factors can be expressed in the same plasmid as the oriP EBNA 7 factor for sustained transgene expression Over time these episomal plasmids are naturally lost at a rate of 5 per cell cycle division due to plasmid dilution promoter silencing and vector replication errors Since most of the plasmid is lost over time cell lines can then be established without any risk of genomic integration or alterations SBI has developed an enhanced version of the oriP EBNA1 technology called the Enhanced Episomal Vector EEV platform This new te
13. mercial products without prior written consent of SBI e This Product should be used in accordance with the NIH guidelines developed for recombinant DNA and genetic research Purchase of the product does not grant any rights or license for use other than those explicitly listed in this Licensing and Warranty Statement 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 SBI disclaims any and all responsibility for injury or damage which may be caused by the failure of the buyer or any other person to use the Product in accordance with the terms and conditions outlined herein Limited Warranty SBI warrants that the Product meets the specifications described in this manual If it is proven to the satisfaction of SBI that the Product fails to meet these specifications SBI will replace the Product or provide the purchaser with a credit This limited warranty shall not extend to anyone other than the original purchaser of the Product Notice of nonconforming products must be made to SBI within 30 days of receipt of the Product SBI s liability is expressly limited to replacement of Product or a credit limited to the actual purchase price SBI 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 w
14. one format no special plasmid production requirements e Nonviral non integrating technology e Persistent sustained transgene expression e Powerful CAGs promoter for constitutive high level transgene expression in all mammalian cell types including primary and stem cells e Tight inducible Cumate Switch format available 888 266 5066 Toll Free 650 968 2200 outside US Page 3 System Biosciences SBI User Manual EEV Cloning Vector Formats SBI has built two formats of EEV cloning and expression vectors to build your own constructs The first features a potent CAGs promoter driving constitutive transgene expression Cat EEV600A 1 The second is an inducible EEV expression vector that features the ultra tight cumate inducible system in an all in one format catalog EEV610A 1 Both vector formats feature a Multiple Cloning Site MCS to insert a cDNA microRNA etc sequence into the EEV vector as well as the EBV oriP origin and the EBNA1 factor for propagating the episome for sustained transgene expression The available cloning and expression EEV vector formats are shown below ColE1 ori CAGs MCS cat EEV600A 1 EBV 10212 bp ori CuO AmpR Promoter CuO MCS cat EEV610A 1 EBV ori 14148 bp EBNA1 Page 4 ver 1 012915 www systembio com EEV Episomal Vector System Cat s EEV6xxA 1 I EEV Reporters and Sample Data A Constitutive and Inducible EEV Reporter Consiruct Maps SBI has built EE
15. st mice n 3 by using 5 ug plasmid and the HDD injection procedure on Day 0 The water soluble version of the inducer cumate compound cat QM150A 1 was injected by IP Page 10 ver 1 012915 www systembio com EEV Episomal Vector System Cat s EEV6xxA 1 1 5 mg per animal to induce EEV expression The mice were then imaged for luminescence activity through full body scans and liver expression levels quantitated from Day 2 through Day 10 The experimental setup is depicted below Day 0 Day 1 Day 2 10 t t t EEV Cumate LUC Cumate Luciferase imaging inducible vector EEV605A 1 1 5 mg IP 15 sec exposure 5 ug mouse injection Hydrodynamic injection Am CuO Promoter Cumate yt ji yA ICH CHC HACOH CuO GFP Luc cat EEV605A 1 The controls for the experiments included a naive no plasmid DNA control as well as EEV605A 1 injected mice n 3 without the addition of cumate The results for the Day 2 measurements are shown in the next figure Luciferase Intensity 24 h Cumate 120000 4000 7 EZ lt a 0 Naive EEV605A 1 EEV605A 1 no Cumate Cumate 5 1000004 3 Sample Mouse Image Data g oe EEV605A 1 E Naive Cumate 60000 a x lt aci 888 266 5066 Toll Free 650 968 2200 outside US Page 11 System Biosciences SBI User Manual Product Information and Protocols Product Format Storage Stability and Availability EEV plasmids are shipped at a concentration of 0 5 u
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