FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2104410020> ?p ?o ?g. }

• W2104410020 endingPage "840" @default.
• W2104410020 startingPage "839" @default.
• W2104410020 abstract "BioTechniquesVol. 49, No. 5 Application Forum - Sponsored PaperOpen AccessCyto-Tracers™: Novel lentiviral-based molecular imaging toolsMaggie Fong, Jake Lesnik, Gang Li, Travis J. Antes & Biao LuMaggie FongSystem Biosciences (SBI), 1616 N. Shoreline Blvd., Mountain View, CA, 94043, USASearch for more papers by this author, Jake LesnikSystem Biosciences (SBI), 1616 N. Shoreline Blvd., Mountain View, CA, 94043, USASearch for more papers by this author, Gang LiSystem Biosciences (SBI), 1616 N. Shoreline Blvd., Mountain View, CA, 94043, USASearch for more papers by this author, Travis J. AntesSystem Biosciences (SBI), 1616 N. Shoreline Blvd., Mountain View, CA, 94043, USASearch for more papers by this author & Biao Lu*To whom correspondence should be addressed. E-mail: E-mail Address: blu@systembio.comSystem Biosciences (SBI), 1616 N. Shoreline Blvd., Mountain View, CA, 94043, USASearch for more papers by this authorPublished Online:3 Apr 2018https://doi.org/10.2144/000113553AboutSectionsPDF/EPUB ToolsAdd to favoritesDownload CitationsTrack Citations ShareShare onFacebookTwitterLinkedInReddit IntroductionFluorescent protein technology has revolutionized cell biology by permitting visualization of a wide range of molecular events within living cells (1). With the continued expansion of stem cell research and developmental biology, there is greater demand for tools to perform real-time monitoring of protein expression and dynamics during reprogramming and lineage commitment. Currently, most plasmid-based technologies in this class are ineffective due to poor transfection efficiencies and narrow tissue expression capabilities (1,2). To overcome these limitations, System Biosciences (SBI) has created a line of lentiviral-based Cyto-Tracers™ (Figure 1A) for effective transduction and stable expression of fluorescent fusion proteins in any mammalian system, including dividing/nondividing cells or whole-model organisms. Using Cyto-Tracers, cellular structures, such as nuclei and mitochondria, can be visualized in a spatial and temporal manner by fluorescence microscopy. In addition, SBI's Cyto-Tracers enable researchers to monitor the dynamic movement of a target protein in relation to a given subcellular compartment by fusing the target protein of interest with different fluorescent proteins. In this report, we present the new Cyto-Tracers product line and demonstrate the powerful and unique applications for their use to gain mechanistic insights into cellular processes that otherwise cannot be performed using standard biochemical or immunological assays.Figure 1. Construction and expression of Cyto-Tracers.Vector configuration and expression of fluorescent fusion proteins using Cyto-Tracers (A). Transient transfection of HEK293 cells shows the specific GFP lighting of various intracellular organelles or structures (A and B). Viral transduction of human fibroblastoma HT1080 cells with CD63-Cyto-Tracer GFP fusion construct shows the formation of intracellular vesicles and subsequent secretion of the exosomes (C).Materials and methodsConstruction of Cyto-TracersEach Cyto-Tracer construct encodes a fusion protein consisting of a unique targeting peptide and either a copGFP or RFP protein sequence (Figure 1A). The fused targeting peptide directs the fluorescent protein to the appropriate subcellular location or even into secretory vesicles like exosomes. SBI also offers an untagged vector for creating customized fusion constructs that are not currently available. Making the fusions with a small, monomeric fluorescent protein sequence is key to allow the appropriate targeting of the protein of interest with minimal disruption. We have chosen two bright monomeric fluorescent proteins to include in the Cyto-Tracer lentivectors for this purpose (CopGFP and ruby RFP).Results and discussionHow Cyto-Tracers workThe newly synthesized proteins have intrinsic sequences and structures, termed “signal peptides” or “address tags,” which govern their transport and localization in the cell. We have built a collection of Cyto-Tracers that have fusions of specific protein-targeting sequences with fluorescent proteins that can highlight various intracellular compartments, organelles, structures (Figure 1B), or extracellular secreted vesicles such as exosomes (Figure 1C). In addition, some of our Cyto-Tracers are able to capture a number of critical biological events with additional natural or artificial sensor sequences, such as BAX-GFP or luciferase circularized with a caspase-3 substrate peptide. The migration of BAX-GFP from cytosol to mitochondria or the activation of the inactive cyclic luciferase upon cell apoptosis allows a real-time monitoring of cell death (3).Performing the transient transfection experiment with Cyto-TracersThe Cyto-Tracers plasmid DNA can be transfected into target cells for use in pilot experiments. As shown in Figure 1, A and B, human HEK293 cells were transfected with either intracellular compartment or organelle-directed CytoTracers vector DNA. Following transfection, the Cyto-Tracers expressed fusion-GFP proteins and within 24 h the cellular compartments, organelles, or structures of the transfected HEK293 cells were specifically marked with copGFP fluorescence as directed by the particular fusion expressed.Performing transduction experiments with Cyto-TracersThe lentiviral-based Cyto-Tracers are the most effective vehicles for transduction under various conditions, including hard-to-transfect mammalian cell lines, whole-animal models, and stem cells. As shown in Figure 1C, HT1080 cells stably transduced and expressing the CD63-Cyto-Tracer (an exosome marker) specifically highlighted intracellular secretory vesicles and a number of the secreted exosomes can also be observed in the living HT1080 cells. The results are glowing exosome vesicles to allow for tracking studies.Lighting up pluripotent stem cells with Cyto-TracersPseudotyped lentiviruses are effective tools to transduce embryonic stem (ES) and induced pluripotent stem (iPS) cells. However, expression of transgenes in stem cells varies in a promoter-dependent manner (2). Consistent with previous reports (2), we observed the inactivation of CMV-driven GFP transgenes in human H9 ES cells (Figure 2, left panel). In contrast, murine stem cell virus promoter (MSCV)—driven GFP expresses at high levels in iPS cells (Figure 2, right panel). Therefore, the MSCV promoter is particularly useful for driving Cyto-Tracer transgene expression in stem cells to enable powerful tracking of protein dynamics.Figure 2. Promoter activity comparisons in stem cells.CMV-driven CytoTracer GFP is selectively inactivated in human H9 ES cells (white circle, left panel), but not in the surrounding mouse embryonic fibroblast feeder cells (white arrows, left panel), when both cells are simultaneously transduced. In contrast, the MSCV-driven Cyto-Tracer GFP lentivirus can be expressed robustly in human induced pluripotent stem cells (right panels).ConclusionsIn response to a growing demand for reliable and high-throughput imaging tools, we have developed a lentiviral-based Cyto-Tracers platform that can accurately capture various molecular events in living cells. Cyto-Tracer constructs combine the lentiviral delivery system with robust fluorescent fusion protein technologies to enable long-term and in-depth studies in virtually any cell type. For reliable expression in stem cells, our MSCV-driven Cyto-Tracers will meet the expanding need for stem cell researchers. More information on Cyto-Tracers can be found online at www.systembio.com/ cyto-tracers, or call SBI at 1 (650) 968-2200.AcknowledgementsThis work is supported by a Small Business Innovation Research grant (1R43GM087116-01) from the National Institutes of Health. We thank Joseph Huang for critical comments.References1. Muzzey, D and A. van Oudenaarden. 2009. Quantitative time-lapse fluorescence microscopy in single cells. Annu Rev Cell Dev Biol. 25:301–27.Crossref, Medline, CAS, Google Scholar2. Xia, XF, YS Zhang, CR Zieth, and SC. Zhang. 2007. Transgene delivered by lentiviral vector are suppressed in a promoter-dependent manner. Stem Cells Dev. 16:167–176.Crossref, Medline, CAS, Google Scholar3. Kanno, A, Y Yamanaka, H Hirano, Y Umezawa, and T. Ozawa. 2007. Cyclic luciferase for real-time sensing of caspase-3 activities in living mammals. Angew Chem Int Ed Engl. 46(40):7595–9.Crossref, Medline, CAS, Google ScholarFiguresReferencesRelatedDetailsCited ByDevelopment of exosome surface display technology in living human cellsBiochemical and Biophysical Research Communications, Vol. 472, No. 1Fluorescent protein biosensors applied to microphysiological systems19 May 2015 | Experimental Biology and Medicine, Vol. 240, No. 6 Vol. 49, No. 5 Follow us on social media for the latest updates Metrics History Published online 3 April 2018 Published in print November 2010 Information© 2010 Future Science LtdAcknowledgementsThis work is supported by a Small Business Innovation Research grant (1R43GM087116-01) from the National Institutes of Health. We thank Joseph Huang for critical comments.PDF download" @default.
• W2104410020 created "2016-06-24" @default.
• W2104410020 creator A5031085364 @default.
• W2104410020 creator A5074942308 @default.
• W2104410020 creator A5081560570 @default.
• W2104410020 creator A5088386954 @default.
• W2104410020 creator A5088489594 @default.
• W2104410020 date "2010-11-01" @default.
• W2104410020 modified "2023-09-25" @default.
• W2104410020 title "Cyto-Tracers™: Novel lentiviral-based molecular imaging tools" @default.
• W2104410020 cites W2040479298 @default.
• W2104410020 cites W2131320542 @default.
• W2104410020 cites W2141594707 @default.
• W2104410020 doi "https://doi.org/10.2144/000113553" @default.
• W2104410020 hasPublicationYear "2010" @default.
• W2104410020 type Work @default.
• W2104410020 sameAs 2104410020 @default.
• W2104410020 citedByCount "2" @default.
• W2104410020 countsByYear W21044100202015 @default.
• W2104410020 countsByYear W21044100202016 @default.
• W2104410020 crossrefType "journal-article" @default.
• W2104410020 hasAuthorship W2104410020A5031085364 @default.
• W2104410020 hasAuthorship W2104410020A5074942308 @default.
• W2104410020 hasAuthorship W2104410020A5081560570 @default.
• W2104410020 hasAuthorship W2104410020A5088386954 @default.
• W2104410020 hasAuthorship W2104410020A5088489594 @default.
• W2104410020 hasBestOaLocation W21044100201 @default.
• W2104410020 hasConcept C136339569 @default.
• W2104410020 hasConcept C207001950 @default.
• W2104410020 hasConcept C54355233 @default.
• W2104410020 hasConcept C70721500 @default.
• W2104410020 hasConcept C86803240 @default.
• W2104410020 hasConceptScore W2104410020C136339569 @default.
• W2104410020 hasConceptScore W2104410020C207001950 @default.
• W2104410020 hasConceptScore W2104410020C54355233 @default.
• W2104410020 hasConceptScore W2104410020C70721500 @default.
• W2104410020 hasConceptScore W2104410020C86803240 @default.
• W2104410020 hasIssue "5" @default.
• W2104410020 hasLocation W21044100201 @default.
• W2104410020 hasLocation W21044100202 @default.
• W2104410020 hasOpenAccess W2104410020 @default.
• W2104410020 hasPrimaryLocation W21044100201 @default.
• W2104410020 hasRelatedWork W1985413631 @default.
• W2104410020 hasRelatedWork W1990804418 @default.
• W2104410020 hasRelatedWork W1993764875 @default.
• W2104410020 hasRelatedWork W2000584892 @default.
• W2104410020 hasRelatedWork W2037347562 @default.
• W2104410020 hasRelatedWork W2046158694 @default.
• W2104410020 hasRelatedWork W2082860237 @default.
• W2104410020 hasRelatedWork W2130076355 @default.
• W2104410020 hasRelatedWork W2137346826 @default.
• W2104410020 hasRelatedWork W2418774313 @default.
• W2104410020 hasVolume "49" @default.
• W2104410020 isParatext "false" @default.
• W2104410020 isRetracted "false" @default.
• W2104410020 magId "2104410020" @default.
• W2104410020 workType "article" @default.