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Cyto-Tracers GFP fusion proteins for in vivo tracingCat. # CYTOxxxUser ManualSystem Biosciences (SBI)2438 Embarcadero WayPalo Alto, CA 94303Tel:888.266.5066 (Toll Free in US)650.968.2200Fax:650.968.2277E-mail: info@systembio.comWeb: www.systembio.com(ver. 2-051818)Store kit at -20 C on receipt(ver. 2-051818)A limited-use label license covers thisproduct. By use of this product, youaccept the terms and conditions outlinedin the Licensing and Warranty Statementcontained in this user manual.

Cyto-Tracers Cat. # CYTOxxxContentsI.Introduction and Background .2II.Available CytoTracers .5III.Protocol .6A.Transfection of pCT-Vector into Target Cells .6B.Transduction of pCT-Vector into Target Cells .6IV.Examples .7A. GFP fluorescence from Cyto-Tracers with differentsubcellular location-specific tags .7B. pCT-vectors can be either transduced or transfected into cells.8V.Licensing and Warranty .9List of ComponentsEach vial contains 10 µg plasmid DNA in TE buffer.Vials are shipped at room temperature or on blue ice and should bestored at -20 C upon receipt. Properly stored samples are stable for 1year from the date received.888-266-5066 (Toll Free)650-968-2200 (outside US)Page 1

System Biosciences (SBI)I.User ManualIntroduction and BackgroundMolecular trafficking is a dynamic process in eukaryotic cells. TheCyto-Tracers provide a means of live monitoring of molecules inreal-time. SBI has created a line of lentivector-based Cyto-Tracersthat utilize GFP-fusion proteins to mark cellular compartments,organelles, vesicles and structures to enable more long-term andmore in-depth experimentation. Using Cyto-Tracers, cellularstructures can be visualized in real-time by fluorescencemicroscopy without the use of chemical staining. You can alsomonitor the dynamic movement of a protein-of-interest inrelationship to a given subcellular compartment or structure bylabeling the protein-of-interest with separate fluorescent proteins.The Cyto-Tracers are built upon an HIV-based lentiviralexpression system to enable broad tropism for delivery, stableexpression via integration, robust synthesis, and expression byusing appropriate promoter elements. To ensure the expression ofthese constructs in a variety of cell types, constructs are availablewith two extensively-tested promoters. The Murine Stem Cell Viruspromoter (MSCV) is useful for stem cells and hematopoietic celltypes. The Cytomegalovirus promoter (CMV)-driven constructscan be used for other, easier to infect cell types. Cyto-Tracers canbe used in transfections or packaged into virus to create stableGFP tracer cell lines in primary cells, tumor cell lines and stemcells. This allows researchers to perform preliminary plasmid-basedtransfection experiments before proceeding to package lentivirusparticles, which allow long-term and in depth studiesPage 2ver. 2-051818www.systembio.com

Cyto-Tracers Cat. # CYTOxxxVector ComponentsThe components of the pCT-CMV/MSCV-GFP-Fusion-EF1-Purovectors are displayed in the table below.Hybrid RSV-5’LTRpromotercPPT, GAG, LTRsCMV or MSCVGFP-FusionproteinEF1 PromoterPuroWPRE element5’ and 3’Δ-LTRpUC originAmpicillinresistanceSV40 originSV40polyadenylationsignal888-266-5066 (Toll Free)For HIV-based vectors. Provides a highlevel of expression of full-lengthpseudoviral constructs in 293 producercells.Genetic elements necessary for thepackaging, transduction, and stableintegration of the viral expressionconstruct into genomic DNA.Promoter that drives expression of theGFP-fusion protein in the construct. TheMSCV promoter is useful for cells that aredifficult to infect or transfect such as stemcells and hematopoetic cells. The CMVpromoter is useful for easy-to-infect cells.This portion of the vector differsdepending on the CytoTracer you haveordered.Drives expression of the puromycinresistance gene.Providespuromycinresistanceforcreation of stable cell lines fromtransfection, or for selection of infectedcells.Enhances stability and translation of thelentivector-driven tion into the host genome.Ensures high copy replication andmaintenance of the plasmid in E.coli cells.Used for selection in E. coli cells.Provides stable propagation of thelentiviral plasmid in 293TN producer dprocessingofrecombinant transcripts.650-968-2200 (outside US)Page 3

System Biosciences (SBI)User ManualGFP-Fusion Protein CharacteristicsEach construct encodes a fusion protein consisting of anorganelle-specific signaling peptide and the copGFP. Theorganelle-specific signaling peptide directs the copGFP to theappropriate subcellular location. SBI also offers a GFP untaggedvector for researchers who would like to create their own tag forsubcellular locations not currently available.copGFP is a novel natural green monomeric GFP-like proteinderived from copepod (Pontellina sp.). The copGFP has optimizedhuman codons for a high level of expression of the fluorescentprotein. CopGFP is a non-toxic, non-aggregating protein with fastprotein maturation, high stability at a wide range of pH (pH4-12),and does not require any additional cofactors or substrates. ThecopGFP protein has very bright fluorescence that exceeds at least1.3 times the brightness of EGFP, the widely used Aequoreavictoria GFP mutant. The copGFP protein emits greenfluorescence with the following characteristics:excitation wavelength max 482 nmemission wavelength max 502 nmDue to its exceptional properties, copGFP is an excellentfluorescent marker that can be used instead of EGFP formonitoring molecular dynamics.Page 4ver. 2-051818www.systembio.com

Cyto-Tracers II.Cat. # CYTOxxxAvailable T-Mito-GFPTarget LocationPlasma MembraneInner Leaflet umGolgi ApparatusER-Golgi MV-PSD95GFPpCT-Cyto-GFPpCT-Cyto-RFP888-266-5066 (Toll eusCaspase3/7activationCytosol toMitochondriaCytosol to Nucleus/Cell olicPeptide TagNeuromodulinFarnesylation signalCytochrome COxidase VIIIER targetingconsensusGalactosyltransferaseSecretory consensuspeptideCathepsin BRhoBPeroxisomalConsensus SignalLC3CD63 Tetraspaninβ-actinα-tubulinMicrotubuleassociated protein 4(MAP4)H2BCyclic inactiveluciferaseBAXβ-Catenin 00 (outside US)Page 5

System Biosciences (SBI)pCT-Cyto-GFP-LucIII.User ManualCompartmentGFP and LuciferaseControlGFP and Luciferase(Untagged)ProtocolA. Transfection of pCT-Vector into Target CellsThe pCT vectors can be transfected into target cells for use in pilotexperiments or for use in cells that are easy to transfect. Werecommend using SBI’s PureFection nanotechnology-basedtransfection reagent according to the ction protocol ver2.pdfB. Transduction of pCT-Vector into Target CellsFor cells that are more difficult to transfect, such as stem cells,primary cells, or hematopoietic cells, or to create stable GFPtracer cell lines, we recommend packaging the pCT-vector intovirus and then transducing the target cells with the virus. For aprotocol and list of necessary supplies and reagents, please referto the Lentivector Expression Systems ogy/deliverysystems/ppack/?id product 11#product 8 tab 1 4For detailed plasmid maps for each of the Cytotracer mattech@systembio.com.Page 6ver. 2-051818www.systembio.com

Cyto-Tracers IV.Cat. # CYTOxxxExamplesA. GFP fluorescence from Cyto-Tracers withdifferent subcellular location-specific tags888-266-5066 (Toll Free)650-968-2200 (outside US)Page 7

System Biosciences (SBI)User ManualB. pCT-vectors can be either transduced ortransfected into cellsPage 8ver. 2-051818www.systembio.com

Cyto-Tracers V.Cat. # CYTOxxxLicensing and WarrantyLimited Use LicenseUse of the Cyto-Tracers (i.e., the “Product”) is subject to the followingterms and conditions. If the terms and conditions are not acceptable,return all components of the Product to System Biosciences (SBI) within 7calendar days. Purchase and use of any part of the Product constitutesacceptance of the above terms.The purchaser of the Product is granted a limited license to use theProduct under the following terms and conditions:The Product shall be used by the purchaser for internal researchpurposes only. The Product is expressly not designed, intended, orwarranted for use in humans or for therapeutic or diagnostic use.The Product may not be resold, modified for resale, or used tomanufacture commercial products without prior written consent of SBI.This Product should be used in accordance with the NIH guidelinesdeveloped for recombinant DNA and genetic research.HIV Vector SystemThis product is for non-clinical research use only. Use of this Product toproduce products for resale or for any diagnostic, therapeutic, clinical,veterinary, or food purpose is prohibited. In order to obtain a license touse this Product for these commercial purposes, contact the Office ofResearch and Technology Ventures at the Dana-Farber Cancer Institute,Inc. in Boston, Massachusetts, USA. This Product or the use of thisProduct is covered by U.S. Patents Nos. 5,665,577 and 5,981,276 (andforeign equivalents) owned by the Dana-Farber Cancer Institute, Inc.WPRE TechnologySBI has a license to sell the Product containing WPRE, under the termsdescribed below. Any use of the WPRE outside of SBI’s Product or theProducts’ intended use requires a license as detailed below. Before usingthe Product containing WPRE, please read the following licenseagreement. If you do not agree to be bound by its terms, contact SBIwithin 10 days for authorization to return the unused Product containingWPRE and to receive a full credit.The WPRE technology is covered by patents issued to The SalkInstitute for Biological Studies.888-266-5066 (Toll Free)650-968-2200 (outside US)Page 9

System Biosciences (SBI)User ManualSBI grants you a non-exclusive license to use the enclosed Productcontaining WPRE in its entirety for its intended use. The Productcontaining WPRE is being transferred to you in furtherance of, andreliance on, such license. Any use of WPRE outside of SBI’s Productor the Product’s intended use requires a license from the Salk Institutefor Biological Studies.This license agreement is effective until terminated. You mayterminate it at any time by destroying all Products containing WPRE inyour control. It will also terminate automatically if you fail to complywith the terms and conditions of the license agreement. You shall,upon termination of the license agreement, destroy all Productscontaining WPRE in you control, and so notify SBI in writing.This License shall be governed in its interpretation and enforcementby the laws of California.Contact for WPRE Licensing: The Salk Institute for Biological Studies,10010 North Torrey Pines Road, La Jolla, CA 92037; Attn: Office forTechnology Management; Phone: (858) 435-4100 extension 1275;Fax: (858) 450-0509.CMV PromoterThe CMV promoter is covered under U.S. Patents 5,168,062 and5,385,839 and its use is permitted for research purposes only. Anyother use of the CMV promoter requires a license from the Universityof Iowa Research Foundation, 214 Technology Innovation Center,Iowa City, IA 52242CopGFP ControlThis product contains a proprietary nucleic acid coding for aproprietary fluorescent protein(s) intended to be used for researchpurposes only. Any use of the proprietary nucleic acids other than forresearch use is strictly prohibited.USE IN ANY OTHERAPPLICATION REQUIRES A LICENSE FROM EVROGEN. To obtainsuch a license, please contact Evrogen at license@evrogen.com.SBI has pending patent applications related to the Product. Forinformation concerning licenses for commercial use, contact SBI.Purchase of the product does not grant any rights or license for useother than those explicitly listed in this Licensing and WarrantyStatement. Use of the Product for any use other than describedexpressly herein may be covered by patents or subject to rights otherthan those mentioned. SBI disclaims any and all responsibility forinjury or damage which may be caused by the failure of the buyer orPage 10ver. 2-051818www.systembio.com