XStamp Exosome Addressing User Manual
Contents
1. or equivalent producer cells a 18 24 hours prior to transfection seed 7 0 8 0 x10 293T cells per 150mm cell culture plate in standard growth media w o antibiotics Cells should be 80 confluent by next day 888 266 5066 Toll Free 650 968 2200 outside US Page 13 System Biosciences SBI User Manual b During transfection day mix 45 ul of pPACKH1 packaging plasmid mix as provided in the LentiStarter 2 0 Kit and 4 5 yg of XStamp lentivector in 1 6 ml of serum free DMEM by pipetting c Add 55 ul PureFection into the same tube Vortex for 10 seconds Note If using other transfection reagents e g Lipofectamine 2000 please follow suggested guidelines for 150mm plates d Incubate mixture at room temperature for 15 minutes e Add mixture drop wise to the dish and swirl to disperse evenly throughout the plates f Change the medium 12 hours or next day after transfection g At 48 hours and 72 hours after transfection collect the medium which now contains pseudoviral particles into a 50 ml sterile capped conical centrifuge tube Centrifuge at 3000 x g for 15 minutes at room temperature to pellet cell debris Transfer the viral supernatant into a new tube Caution You are working with infectious pseudoviral particles at this stage Please follow the recommended guidelines for working with BSL 2 biosafety agents 2 Concentration of Pseudoviral Particles The PEG it Virus Precipitation Solution i2. A Varming K J rgensen M Diagnostic and prognostic potential of extracellular vesicles in peripheral blood Clin Ther 2014 Jun 1 36 6 830 46 doi 10 1016 j clinthera 2014 05 008 PubMed PMID 24952934 Zhang L Wrana JL The emerging role of exosomes in Wnt secretion and transport Curr Opin Genet Dev 2014 Aug 27 14 9 doi 10 1016 j gde 2014 03 006 Epub 2014 May 8 Review PubMed PMID 24791688 Drake RR Kislinger T The proteomics of prostate cancer exosomes Expert Rev Proteomics 2014 Apr 11 2 167 77 doi 10 1586 14789450 2014 890894 Epub 2014 Feb 25 PubMed PMID 24564711 Soldevilla B Rodriguez M San Millan C Garcia V Fernandez Peria ez R Gil Calder n B Martin P Garcia Grande A Silva J Bonilla F Dominguez G Tumor derived exosomes are enriched in ANp73 which promotes oncogenic potential in acceptor cells and correlates with patient survival Hum Mol Genet 2014 Jan Page 20 ver 1 150804 www systembio com XStamp Exosome Display Technology Cat s XSTPxxxPA 1 15 23 2 467 78 doi 10 1093 hmg ddt437 Epub 2013 Sep 18 PubMed PMID 24067531 Camacho L Guerrero P Marchetti D MicroRNA and protein profiling of brain metastasis competent cell derived exosomes PLoS One 2013 Sep 16 8 9 e73790 doi 10 1371 journal pone 0073790 eCollection 2013 PubMed PMID 24066071 PubMed Central PMCID PMC3774795 Raimondo F Morosi L Corbetta S Chinello C Brambilla P Della Mina P Villa A Albo G Battaglia C Bosar
3. 4 a Mix 5 uL PureFection reagent 2 5 ug XStamp Lentivector and 200 uL serum free media in sterile 1 5 mL Eppendorf tube b Vortex briefly and incubate at room temperature for 15 minutes c Add entire volume to 6 well of cells in a total volume of 2 3 ml media Change media after 24 hours Isolate exosomes in 48 96 hour window post transfection Page 10 ver 1 150804 www systembio com XStamp Exosome Display Technology Cat s XSTPxxxPA 1 Isolation of XStamp Exosomes and Addition to Target Cells 1 Remove cell culture media and place in 15 mL or 50 mL centrifuge tube Spin centrifuge tubes at 3 000 x g for 30 minutes at room temperature or 4 C to get rid of cellular debris Transfer the supernatant to a new tube Add ExoQuick TC at 1 5 the volume of cell culture media Mix by inversion and incubate at 4 C overnight Spin centrifuge tubes at 1500 x g for 30 minutes at room temperature or 4 C temperature does not affect exosome yield Discard supernatant and resuspend exosome containing pellet in 100 uL PBS Measure exosome yield using A280 on Nanodrop Adjust concentration to 1 ug uL Add exosomes to cell culture dish containing target cells For target cells gt 1 5x10 5 cells in a 6 well plate format 250 ug exosomes is sufficient The number of exosomes required to discern effects in target cells may vary by cell type and by the specific phenotype being assayed therefore optimization of specific experiment
4. 7 SBI System Biosciences XStamp Exosome Targeting Technology Cat s XSTPxxPA VA 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 XStamp Exosome Display Technology Cat s XSTPxxxPA 1 Contents l gt MNTPODUCTION sceri ieir i e dienes 1 A XStamp Technology Overview ccccccceeeeeeeseeeeeseeeees 1 B Uses of XStamp Technology ccceeceeeeeeeeeeeeeeeneeeeeees 4 Il XStamp Sample data cccccececceeeeceeeeeeeeceeeeesseeeesseeseeeees 5 A XStamp Transfection Protocol 0 ccccecceeeeeeeeseeeeeneeees 10 C XStamp Lentiviral Particle Production c ccccsseeeeeses 12 B Concentration of Pseudoviral Particles eeeeeee 14 C Transduction of Pseudoviral Particles into Target Cells 15 D Shipping and Storage Conditions c ccccceeeeeeeeeeees 17 E Related Prodqucts cceccceeeeeeeeeeeeeeeeeeeeeeeeeeseeeeeeeeeeaees 17 III Frequently Asked Questions 0 ceeceeeeeeeeeeteeeeeeeeeeneeees 18 IV ReiCrenCes i 08 cata aie ites en eee eee 18 V Technical SUP POM mriisi a oiaren n oirinn tineo atrau enaus 21 VI Licensing and Warranty information s e 22 I Introduction A XStamp Technology Overview Exosomes are nanosized membrane vesicles secreted by mos
5. P721PA 1 and the XStamp BHP1 construct catalog XSTP722PA 1 were transfected separately into mouse MSCs along with SBI s XPack GFP construct catalog XPAK530PA 1 which packages GFP into the interior of exosomes for fluorescent tracking After 48 hours the exosomes were collected using ultracentrifugation and then quantitated for exosome protein levels Equal amounts 100 ug of Control No XStamp XStamp NCAM or XStamp BHP1 MSC exosomes all labeled with XPack GFP were added to Neuro2a neuroblastoma cells in culture The neurons were imaged for phase and GFP signals after a 24 hour incubation with the various exosomes The cells were imaged after 24 hours for uptake of the XStamp NCAM coated and BHP1 coated exosomes to deliver the XPack GFP exosomes Together they are known as the Pack and Stamp system Page 8 ver 1 150804 www systembio com XStamp Exosome Display Technology Cat s XSTPxxxPA 1 No XStam XStamp NCAM XStamp BHP1 GFP RAPS S a A Oe MSC exosomes with XPack GFP XStamps added to primary neurons cells in culture imaged after 24 hours post exosome addition The Neuro2a neuroblastoma cells that are NCAM positive took up the XStamp NCAM and XStamp BHP1 exosomes at a much higher rate than control XStamp exosomes NOTE ExoQuick TC and Exo FBS are not provided with the XStamp vectors and can be purchased separately The following ExoQuick TC products are recommended for exosome concentration prior to a
6. al conditions may be needed B Cloning of Proteins into XStamp MCS 1 To clone fusions in frame note that the XStamp lentivectors have a 5 leader sequence to facilitate secretion and a 3 C1C2 XStamp domain for exosome display If your ligand of interest already has an endogenous signal sequence peptide then use the Xbal site upstream of the built in leader sequence and replace with cDNA of your choice 888 266 5066 Toll Free 650 968 2200 outside US Page 11 System Biosciences SBI User Manual 2 It may be necessary to add 1 or 2 bases to the 3 end of the ORF to generate an in frame fusion In such cases count the number of nucleotides from the start of the MCS to where first nucleotide in the initial codon of the ORF of interest will be inserted and add as many nucleotides as needed to make that number a multiple of 3 We recommend using the free plasmid editor software to design the XStamp fusions and ensure the full ORF from the ATG in the leader through cDNA into C1C2 XStamp domain is intact Download the free plasmid editor software here http www systembio com support resources online tools For technical support email tech systembio com to request the XStamp cloning lentivector plasmid editor annotated sequence file If you need clone design assistance email tech systembio com as well C XStamp Lentiviral Particle Production For researchers looking for sustained long term expression of the XStam
7. ay any protein of choice on their surfaces These exosomes can then be used to efficiently program specific delivery to cells that have a cognate receptor allowing for more targeted exosome cargo delivery both in vitro and in vivo Surface display on exosomes can also be used to boost vaccine generation and engineer other drug screening applications Increasing vaccine potency through exosome antigen targeting Hartman ZC Wei J Glass OK Guo H Lei G Yang XY Osada T Hobeika A Delcayre A Le Pecq JB Morse MA Clay TM Lyerly HK Vaccine 2011 Nov 21 29 50 9361 7 Exosome nanovesicles displaying G protein coupled receptors for drug discovery Estelles A Sperinde J Roulon T Aguilar B Bonner C LePecg JB Delcayre A Int J Nanomedicine 2007 2 4 751 60 Exosomes as novel therapeutic nanodevices Delcayre A Le Pecq JB Curr Opin Mol Ther 2006 Feb 8 1 31 8 Review Page 4 ver 1 150804 www systembio com XStamp Exosome Display Technology Cat s XSTPxxxPA 1 XStamp Sample data A XStamp Motilin Motilin is a 22 amino acid polypeptide hormone in the motilin family that in humans is encoded by the MLN gene Motilin is secreted by endocrine M cells that are numerous in crypts of the small intestine especially in the duodenum and jejunum The Motilin receptor is a G protein coupled receptor that binds motilin and is exclusively expressed in the intestine To test the XStamp Motilin construct catalog XSTP720PA 1 it was transfecte
8. d into HEK293 cells and after 48 hours the exosomes were collected using ExoQuick TC The next day the XStamp Motilin exosomes were Exo Fected with a Texas Red labeled siRNA to monitor exosome docking and delivery The transfected XStamp Motilin exosomes were then added to MDA MB 231 Breast Cancer Cells motilin receptor negative and to HT 29 Colon Cancer Cells motilin receptor positive The cells were imaged after 24 hours for uptake of the Texas Red labeled siRNA delivery from the XStamped exosomes The HT 29 colon cancer cells that are motilin receptor positive took up the XStamp Motilin exosomes at a much higher rate than the MDA MB 231 Breast Cancer motilin receptor negative cells 888 266 5066 Toll Free 650 968 2200 outside US Page 5 System Biosciences SBI User Manual Produce Motilin XStamp ove 4 HEK293 Exosomes o E Exo Fect with 4 Texas Red eae labeled siRNA Add labeled XStamp Motilin Exosomes to Breast cancer or Colon Cancer Cells o MDA MB 231 Breast Cancer Cells HT 29 Colon Cancer Cells RFP filter set RFP filter set B XStamp NCAM and XStamp BHP1 The Neural Cell Adhesion Molecule 1 NCAM gene encodes a cell adhesion protein which is a member of the immunoglobulin superfamily The encoded protein is involved in cell to cell interactions as well as cell matrix interactions during development and differentiation The encoded protein has been shown to be involved in development
9. ddition to target cells Cat Description Size EXOTC10A 1 ExoQuick TC for Tissue 10 ml Culture Media and Urine EXOTC50A 1 ExoQuick TC for Tissue 50 ml Culture Media and Urine EXO FBSHI Exosome depleted FBS 50 mi 50A 1 media supplement Heat Inactivated IMPORTANT NOTE Be sure to culture your exosome producer cell lines in media that does not contain standard 888 266 5066 Toll Free 650 968 2200 outside US Page 9 System Biosciences SBI User Manual FBS There are high levels of bovine exosomes present in FBS Instead use SBI s Exo FBS Heat Inactivated Exosome depleted FBS Media Supplement cat EXO FBSHI 50A 1 in place of standard FBS media supplements A XStamp Transfection Protocol Transfection of Exosome Producer Cells 1 Seed exosome producer cells in culture dish of choice to reach 70 80 confluency after 24 hours using media compatible with the cells of choice Because standard FBS contains high levels of bovine exosomes be sure to use SBI s Exosome depleted FBS Media Supplement to ensure that exosomes isolated after cell transfection are not contaminated by bovine exosomes Return cells to incubator 24 hours later mix XStamp vector with transfection reagent of choice and follow appropriate protocol to achieve transfection of target cells An example transfection reaction using SBI s PureFection Cat LV750A 1 in a 6 well plate of cells at 70 80 confluency 3
10. i S Magni F Pitto M Differential protein profiling of renal cell carcinoma urinary exosomes Mol Biosyst 2013 Jun 9 6 1220 33 doi 10 1039 c3mb25582d Epub 2013 Mar 19 PubMed PMID 23511837 de Jong OG Verhaar MC Chen Y Vader P Gremmels H Posthuma G Schiffelers RM Gucek M van Balkom BW Cellular stress conditions are reflected in theprotein and RNA content of endothelial cell derived exosomes J Extracell Vesicles 2012 Apr 1631 doi 10 3402 jev v1i0 18396 eCollection 2012 PubMed PMID 24009886 PubMed Central PMCID PMC3760650 V Technical Support For more information about SBI products and to download manuals in PDF format please visit our web site http www systembio com For additional information or technical assistance please call or email us at System Biosciences SBI 265 North Whisman Rd SBI Mountain View CA 94043 888 266 5066 Toll Free 650 968 2200 outside US Page 21 System Biosciences SBI User Manual Phone 650 968 2200 888 266 5066 Toll Free Fax 650 968 2277 E mails General Information info systembio com Technical Support tech systembio com Ordering Information orders systembio com VI Licensing and Warranty information Limited Use License Use of the XStamp 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 ca
11. in 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 warranty 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 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 23
12. lendar 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 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 commercial 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 e This Product is a Patented technology under US Patent 7 704 964 licensed from ExoThera LLC e Uses of the technology for commercial purposes requires a license from SBI through ExoThera LLC Page 22 ver 1 150804 www systembio com XStamp Exosome Display Technology Cat s XSTPxxxPA 1 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 here
13. m 2 x 150mm plates 20ml per plate this would be approximately 80ml of media You would resuspend the resulting pellet in 80 160 pl of 1X PBS or DMEM 6 Aliquot in cryogenic vials and store at 80 C until ready for use 7 The resulting pseudoviral particles can be accurately titered using SBI s UltraRapid Global Titering Kit Cat LV961A 1 http www systembio com lentiviral technology delivery systems ultrarapid overview C Transduction of Pseudoviral Particles into Target Cells For efficient transduction of target cells the negative charges present in the virus envelope protein and the cell surface must be neutralized SBs TransDux reagent provided in the LentiStarter 2 0 Kit is a non toxic proprietory formulation that promotes cell 888 266 5066 Toll Free 650 968 2200 outside US Page 15 System Biosciences SBI User Manual virus contact and subsequent fusion by negating these charges The following protocol can be utilized for delivery of virus to your target cells The following protocol is for infection of target cells in a single well of a 24 well plate if using larger vessels please scale up reagents accordingly Day 1 1 Plate 75 000 cells per well into a single well of a 24 well plate in cell culture medium Make sure that cells are well dispersed and are not clumped together Include wells for negative non infected cells Note If infecting target cells for the first time or an optimal MOI is n
14. n the LentiStarter 2 0 Kit provides a simple and highly effective means to concentrate lentiviral particles PEG it is a formulation of polyethylene glycol optimized for the precipitation of lentiviral based particles The PEG it Virus Precipitation Solution is provided as a 5x solution 1 Transfer supernatant containing virus to a sterile vessel and add 1 volume of cold PEG it Virus Precipitation Solution 4 C to every 4 volumes of virus supernatant Example 5ml PEG it with 20ml viral supernatant Page 14 ver 1 150804 www systembio com XStamp Exosome Display Technology Cat s XSTPxxxPA 1 2 Refrigerate overnight at least 12 hours Viral supernatants mixed with PEG it Virus Precipitation Solution are stable for up to 4 5 days at 4 C 3 Centrifuge supernatant PEG it mixture at 1500 x g for 30 minutes at 4 C After centrifugation the virus particles may appear as a beige or white pellet at the bottom of the vessel 4 Discard the supernatant into a suitable biohazard waste container Spin down residual PEG it solution by centrifugation at 1500 x g for 5 minutes Remove all traces of fluid by aspiration taking great care not to disturb the precipitated lentiviral particles in pellet 5 Resuspend lentiviral pellets in 1 500 to 1 1000 of original volume of pooled virus supernatant using cold sterile Phosphate Buffered Saline PBS or DMEM containing 25mM HEPES buffer at 4 C For example if you performed 2 collections fro
15. of the nervous system The NCAM domains interact with each other during the adhesion process Page 6 ver 1 150804 www systembio com XStamp Exosome Display Technology Cat s XSTPxxxPA 1 TiTa NCAM Mediates Neural Cell Adhesion WAPI The seven domain protein binds to an identical NCAM on the opposing membrane to form cell cell junctions NCAM cpi Signal membrane peptide anchor Homophilic Signaling TRO 20 300 400 50 6 amp 0 U NCAM Clone NCAMs An XStamp NCAM fusion was constructed and incorporated the first 300 amino acids Signal Peptide plus IGc2 domains 1 3 of the mouse NCAM gene translationally fused to the C1C2 XStamp display tag catalog XSTP721PA 1 In parallel a Brain Homing Peptide BHP1 from Organ targeting in vivo using phage display peptide libraries Pasqualini R Ruoslahti E Nature 1996 Mar 28 380 6572 364 6 was fused to the BHP1 XStamp domain to create the XStamp NCAM construct catalog XSTP722PA 1 888 266 5066 Toll Free 650 968 2200 outside US Page 7 System Biosciences SBI User Manual RSV promoter 7 234 HIVILTR 235 414 AmpR 8465 7605 RRE 1076 1308 cPPT 1798 1916 ia areca XSTP721PA 1_mNCAM MSCVmulpromoter 1928 2339 9069 bp SV40 Ori 6271 6417 SV40 EEL poly A s 6131 6262 3 dLTR 5826 6059 mNCAM1 truncated 2345 3227 f WPRE 5164 5754 XStamp 3242 3997 puro 4558 5154 EF1 promoter 4007 4552 To test the XStamp NCAM construct catalog XST
16. osomes e ExoQuick exosome isolation reagents Exo FBS exosome depleted media supplement e Detect and quantitate exosomes with ExoAB ELISA and EXOCET kits e Purify exosome RNA and profile by qPCR with SeraMir Kit e Discover novel exoRNA biomarkers with Exo NGS next gen sequencing services 888 266 5066 Toll Free 650 968 2200 outside US Page 17 System Biosciences SBI User Manual lll Frequently Asked Questions Q How long and in what condition should store exosomes after isolation from an exosome generating cell line After exosomes are isolated with ExoQuick TC the pellet can be stored at 80 C for 1 year After resuspension in PBS it can be stored at 4 C for 2 weeks or 20 C for 3 months Q How many exosomes should I add to my target cells 250 ug of exosomes as determined by A280 on NanoDrop is sufficient to see efficient delivery of XStamp coated exosomes on target cells in a 6 well plate format The number of exosomes required in culture dishes of other size can be scaled up or down proportionally to the difference in total cell number relative to one well of a 6 well plate Example HEK2983T cells 6 well seeding density 400 000 cells 24 well seeding density 100 000 cells 100 000 400 000 1 4 number of cells 250 ug exosomes x 14 62 5 ug exosomes for use in 24 well plate format IV References Hartman ZC Wei J Glass OK Guo H Lei G Yang XY Osada T Hobeika A Delcayre A Le Pecq JB Mo
17. ot known please titrate virus at varying MOls 1 5 10 and 20 etc to optimize transduction using a positive control virus with a fluorescent marker such as SBI s pre packaged positive transduction control Cat CD511VB 1 Day 2 2 Cells should be between 70 80 confluent Aspirate medium from cells 3 Combine culture medium with TransDux to a 1X final concentration For example add 2 5 ul of TransDux to 500 ul culture medium and then transfer to each well If using other types of transduction reagents e g Polybrene please dilute the reagent to a final working concentration of 2 8 pg ml 4 Add XStamp virus at desired MOI to each well and swirl to mix for negative control wells only add media viral transduction reagent Day 3 5 Aspirate off medium and add complete growth medium to cells Page 16 ver 1 150804 www systembio com XStamp Exosome Display Technology Cat s XSTPxxxPA 1 Day 5 7 Virus should be integrated into the host cell genome by this time and should be expressing the XStamp construct for packaging into exosomes D Shipping and Storage Conditions The XStamp lentivectors are shipped on either blue ice 4 C or dry ice and should be stored at 20 C upon arrival Avoid freeze thawing the reagents Shelf life of either product is 1 year after receipt if stored properly E Related Products SBI offers a number of exosome research products You can review them here http Awww systembio com ex
18. p tag Flow cytometry data using cells and exosomes demonstrate the enrichment of MFG E8 on exosomes Figure below MFG E8 CD58 CD81 Cells C1C2 domain Cells and Exosomes s wosoxJ Counts e Tetraspanins MFG E8 i CD63 CD81 CD9 etc Fluorescence intensity Fluorescently labeled antibodies for MFG E8 CD58 and CD81 were used in combination for FACs analysis The CD58 marker is a known cell surface marker that is absent on exosomes CD81 is known to be present both in cells and exosomes The FACs data show that MFG E8 is exclusively detected on exosomes and not present in the cells Page 2 ver 1 150804 www systembio com XStamp Exosome Display Technology Cat s XSTPxxxPA 1 XStamp exosome surface engineering To take advantage of the localization of MFG E8 on exosomes the C1C2 domain XSitamp domain of the protein s gene was cloned into SBI s MSCV MCS EF1 Puro lentivector and a 5 secretion signal SS was placed within the multiple cloning site The protein ligand chosen to program the exosomes to target cells with a cognate receptor is the Exposed Ligand part of the construct A lentivector plasmid general format is shown below Exposed Ligand XStamp Expression Lentivector Replication The XStamp expression cassette is driven by the MSCV promoter that works in most cell lines including primary cells and stem cells There is a built in signal sequence SS next to the multicloning site
19. p construct in their desired cell line the XStamp construct can be transfected into HEK293T producer cells and packaged into pseudo viral particles for infection of a target cell line The following schematic and the protocol that follows shows the lentiviral production process using the XStamp lentiviral vector Page 12 ver 1 150804 www systembio com XStamp Exosome Display Technology Cat s XSTPxxxPA 1 How to Make High Titer Lentivirus Make Your Lentivector Clone cDNA shRNA reporter microRNA etc Construct pPACK Packaging Mix To make lentivirus co transfect your P lentivector plus pPACK packaging mix 4 e Plasmids a using PureFection into 293TN packaging cell line 293TN Producer Cells Wait 48 72 hours collect supernatant and adii combine with PEG it virus concentration Medium Containing solution Next day remove supernatant and Viral Particles 5x PEG it resuspend pellet in sterile PBS Solution fol a Y Concentrate Virus Check Titer using Global UltraRapid Titer Kit using standard cell line ex 293 cells Global Titering Kit Measure Titer by qPCR 5 Combine the appropriate amount of virus with TransDux and infect your target cells Animal Models Primary Cells Human Embryonic H9 Cells Mouse Carotid Human Primary Phase contrast GFP Artery GFP Neurons GFP Workflow for generating high titer lentiviral particles 1 Transfection of XStam lasmids into HEK293T
20. rse MA Clay TM Lyerly HK Increasing vaccine potency through exosome antigen targeting Vaccine 2011 Nov 21 29 50 9361 7 Estelles A Sperinde J Roulon T Aguilar B Bonner C LePecg JB Delcayre A Exosome nanovesicles displaying G protein coupled receptors for drug discovery Int J Nanomedicine 2007 2 4 751 60 Page 18 ver 1 150804 www systembio com XStamp Exosome Display Technology Cat s XSTPxxxPA 1 Delcayre A Le Pecq JB Exosomes as novel therapeutic nanodevices Curr Opin Mol Ther 2006 Feb 8 1 31 8 Review Morse MA Garst J Osada T Khan S Hobeika A Clay TM Valente N Shreeniwas R Sutton MA Delcayre A Hsu DH Le Pecq JB Lyerly HK A phase study of dexosome immunotherapy in patients with advanced non small cell lung cancer J Transl Med 2005 Feb 21 3 1 9 Pirjo Laakkonen and Kirsi Vuorinena Homing peptides as targeted delivery vehicles Integr Biol 2010 2 326 337 Rountree RB et al Exosome targeting of tumor antigens expressed by cancer vaccines can improve antigen immunogenicity and therapeutic efficacy Cancer Res 2011 Aug 1 71 15 5235 44 Gy rgy B Hung ME Breakefield XO Leonard JN Therapeutic applications of extracellular vesicles clinical promise and open questions Annu Rev Pharmacol Toxicol 2015 55 439 64 doi 10 1146 annurev pharmtox 010814 124630 Epub 2014 Oct 3 PubMed PMID 25292428 van der Meel R Fens MH Vader P van Solinge WW Eniola Adefeso O Schiffelers RM Extracell
21. t cell types in vivo and in vitro They are produced by the inward budding of multivesicular bodies MVBs and subsequently released from the cell into the microenvironment following the fusion of MVBs with the plasma membrane Exosomes are extracellular nanoshuttles that facilitate communication between cells and organs and are found in various biofluids including blood 888 266 5066 Toll Free 650 968 2200 outside US Page 1 System Biosciences SBI User Manual urine amniotic fluid breast milk malignant ascites fluid and cerebrospinal fluid CSF Exosomes contain distinct subsets of RNAs and proteins depending upon the cell type from which they are secreted making them useful for biomarker discovery Additionally their natural function as cell to cell communication vehicles makes them attractive for use as therapeutic shuttles to deliver biological molecules or drugs to target disease cells SBI has developed an exosome surface display system that enables desired protein sequences to be placed efficiently on the surfaces of engineered exosomes called the XStamp technology The XStamp technology is based upon a C terminal fusion of ligands that enables the efficient display of the ligands on the surfaces of secreted exosomes The C1C2 fusion domain is derived from the human MFG E8 protein and has been shown to be abundant and nearly exclusively localized on the surfaces of exosomes This C1C2 domain is what we are terming the XStam
22. ular vesicles as drug delivery systems lessons from the liposome field J Control Release 2014 Dec 10 195 72 85 doi 10 1016 j jconrel 2014 07 049 Epub 2014 Aug 2 Review PubMed PMID 25094032 Coleman BM Hill AF Extracellular vesicles Their role in the packaging and spread of misfolded proteins associated with neurodegenerative diseases Semin Cell Dev Biol 2015 Feb 20 pii S1084 9521 15 00034 8 doi 10 1016 j semcdb 2015 02 007 Epub ahead of print Review PubMed PMID 25704308 888 266 5066 Toll Free 650 968 2200 outside US Page 19 System Biosciences SBI User Manual Yao Y Wei W Sun J Chen L Deng X Ma L Hao S Proteomic analysis of exosomes derived from human lymphoma cells Eur J Med Res 2015 Jan 29 20 1 8 PubMed PMID 25631545 PubMed Central PMCID PMC4329659 Yang J Wei F Schafer C Wong DT Detection of tumor cell specific mRNA and protein in exosome like microvesicles from blood and saliva PLoS One 2014 Nov 14 9 11 e110641 doi 10 1371 journal pone 0110641 eCollection 2014 PubMed PMID 25397880 PubMed Central PMCID PMC4232306 Zhao X Wu Y Duan J Ma Y Shen Z Wei L Cui X Zhang J Xie Y Liu J Quantitative proteomic analysis of exosome protein content changes induced by hepatitis B virus in Huh 7 cells using SILAC labeling and LC MS MS J Proteome Res 2014 Dec 5 13 12 5391 402 doi 10 1021 pr5008703 Epub 2014 Oct 8 PubMed PMID 25265333 Revenfeld AL B k R Nielsen MH Stensballe
23. where the desired cDNA Exposed Ligand is fused to both the upstream SS and the downstream XStamp CiC2 domain sequence The SS leader peptide assists the secretion of the fusion protein and is cleaved off during the surface display process and loading on the surfaces of secreted exosomes If the desired Exposed Ligand already has its own signal sequence there is an Xbal cloning site 5 to the SS sequence in the XStamp lentivector where the cDNA encoding the SS Exposed Ligand can be inserted into the lentivector The XStamp lentivector also features a downstream EF1 Puromycin cassette for selection and stable cell line development The lentivector constructs can be used in transient transfection expression studies as well as packaging the construct into lentivirus to stably transduce cells A more detailed lentivector plasmid map is depicted below 888 266 5066 Toll Free 650 968 2200 outside US Page 3 System Biosciences SBI User Manual RSV promoter 7 234 HIV LTR 235 414 AmpR 7680 6820 RRE 1076 1308 XSTP710PA 1 cPPT 1798 1916 pUC ORI 6675 6002 8284 bp MSCVmul promoter 1928 2339 2340 Xbal 1 Leader 2348 2419 S V40 0ri 5486 5632 SV4O EEL poly A s 5346 5477 3 dLTR 5041 5274 WPRE 4379 4969 2451 BamHI 1 XStamp 2457 3212 EF1 promoter 3222 3767 puro 3773 4369 B Uses of XStamp Technology The XStamp technology allows for cell mediated generation of ready to use exosomes that displ
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