Instructions rLV Ubi –LifeAct ® –TagGFP2 LifeAct ® is a 17 amino acids long fragment of a protein originating from Saccharomyces cerevisiae, which comprises an actin–binding domain. This marker can be used in various eukaryotic cells to stain filamentous actin (F–actin). Used in living cells it is perfectly labeling the highly dynamic F–actin and moreover, does not interfere with cellular processes. Please read the following Application Note for more detailed handling information: Application Note 41 ” Protocol for Lentiviral Transduction of Human Cells“: This AN provides an example protocol for a stable transduction of HT-1080 in μ-Dish 35mm, high . Vector Description Lentiviral vectors derived from the human immunodefi- ciency virus (HIV-1) have become major tools for stable in- tegration of genes in mammalian cells. The most advanta- geous feature of lentiviral vectors is the ability to mediate efficient transduction, integration, and long-term expres- sion into dividing and non-dividing cells, both in vitro and in vivo. The pseudotyped envelope with vesicular stomatitis virus envelope G (VSV-G) protein broadens the target cell range. Lentiviral vectors have now been shown to deliver genes to neurons, lymphocytes and macrophages. Previous retrovirus vectors could not be used for these cell types. Moreover, lentiviral vectors have proven to be effective in transducing brain, liver, muscle, and retina in vivo without any toxicity effects or immune responses. rLV Ubi –LifeAct ® –TagGFP2 comprises LifeAct ® –TagGFP2 fusion protein and can be used for fluorescent labeling of the actin cytoskeleton in various living cells with spe- cial focus on the generation of stable cell lines. TagGFP2 codon usage is optimized for high expression in mam- malian cells, i.e. humanized. Actin-binding domain of the yeast protein Abp140 is fused to the TagGFP2 N-terminus. For strong expression of the fusion protein in mammalian cells the vector contains the human Ubiquitin C (Ubi) pro- moter. Vector Map 5’–LTR 5’ Long terminal repeat PSI RNA packaging signal RRE Rev response element CTS Central termination sequence Ubi Ubiquitin C promoter IRES Internal ribosome entry site PuroR Puromycin Resistance WPRE Woodchuck Hepatitis Posttranscriptional element 3’ SIN–LTR 3’ self-inactivating long terminal repeat Specifications TagGFP2 Fluorescence Ex max 483 nm Em max 506 nm Find more information on www.evrogen.com. Packaging and Storage Amount 100 μl Concentration 1 × 10 7 TU/ml Shipping conditions dry ice Storage conditions -80°C* Shelf life Under proper storage condi- tions as indicated on vial. *Avoid repeated freeze and thaw cycles as this leads to strong decrease in viral titer. rLV Ubi –LifeAct ® –TagGFP2 Page 1 Version 2.1 (2016-06-08)
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Instructions rLVUbi–LifeAct®–TagGFP2
LifeAct® is a 17 amino acids long fragment of a protein originating from Saccharomyces cerevisiae,which comprises an actin–binding domain. This marker can be used in various eukaryotic cellsto stain filamentous actin (F–actin). Used in living cells it is perfectly labeling the highly dynamicF–actin and moreover, does not interfere with cellular processes.
Please read the following Application Note for more detailed handling information:
Application Note 41 ”Protocol for Lentiviral Transduction of Human Cells“: This AN provides an example protocol for astable transduction of HT-1080 in µ-Dish 35mm, high.
Vector Description
Lentiviral vectors derived from the human immunodefi-ciency virus (HIV-1) have become major tools for stable in-tegration of genes in mammalian cells. The most advanta-geous feature of lentiviral vectors is the ability to mediateefficient transduction, integration, and long-term expres-sion into dividing and non-dividing cells, both in vitro andin vivo.
The pseudotyped envelope with vesicular stomatitis virusenvelope G (VSV-G) protein broadens the target cell range.Lentiviral vectors have now been shown to deliver genesto neurons, lymphocytes and macrophages. Previousretrovirus vectors could not be used for these cell types.
Moreover, lentiviral vectors have proven to be effective intransducing brain, liver, muscle, and retina in vivo withoutany toxicity effects or immune responses.
rLVUbi–LifeAct®–TagGFP2 comprises LifeAct®–TagGFP2fusion protein and can be used for fluorescent labelingof the actin cytoskeleton in various living cells with spe-cial focus on the generation of stable cell lines. TagGFP2codon usage is optimized for high expression in mam-malian cells, i.e. humanized. Actin-binding domain of theyeast protein Abp140 is fused to the TagGFP2 N-terminus.
For strong expression of the fusion protein in mammaliancells the vector contains the human Ubiquitin C (Ubi) pro-moter.
Vector Map
5’–LTR 5’ Long terminal repeatPSI RNA packaging signalRRE Rev response elementCTS Central termination sequenceUbi Ubiquitin C promoter
The transduction efficiency of lentiviral particles varies indifferent cell types and therefore, has to be determined inadvance for obtaining the optimal transduction rate. Arange of 0.5–5 MOIs (multiplicity of infection) is used formost cell types. However, up to 20 MOI may be necessaryfor suspension cells.
Note:
Hexadimethrine bromide (Polybrene) enhances trans-duction of most cell types. Some cells, like primaryneurons, are sensitive to hexadimethrine bromide. Donot add hexadimethrine bromide to these types ofcells. If working with a cell type for the first time,only one hexadimethrine control well should be usedto determine cell sensitivity.
Selection of Stable Cells
For selection of stable cells, puromycin needs to be addedto the culture medium 48-72 hours after the transductionprocess. Replace media with fresh puromycin contain-ing medium every 3-4 days until resistant colonies canbe identified. For best results, puromycin needs to betitrated with nontransduced cells. Typical final concentra-tions range from 0.1–10 µg/ml.
Safety Consideration:
Note, that you will be working with samples contain-ing infectious virus. Follow the recommended NIHguidelines for all materials containing BSL–2 organ-isms. Always wear gloves, use filtered tips and workunder a biosafety hood.
Selected References
J. Riedl, A. H. Crevenna, K. Kessenbrock, J. H. Yu, D. Neukirchen, M. Bista, F. Bradke, D. Jenne, T. A. Holak, Z. Werb,M. Sixt, and R. Wedlich-Soldner. Lifeact: a versatile marker to visualize f-actin. Nature Methods, 5(7):605–607, 2008. doi:10.1038/nmeth.1220.
rLVUbi–LifeAct®–TagGFP2 Page 2 Version 2.1 (2016-06-08)
This product is for internal non–commercial research use only. No rights are conveyed to modify or clone the gene encoding fluorescent protein containedin 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: [email protected].
LifeAct® Product and its Variants
Use of these products and product applications are covered by european and foreign patents and patents pending.
All names containing the designation ® are registered with the Office for Harmonization in the Internal Market and the U.S. Patent and Trademark Office.
Not–For–Profit Entities: Orders may be placed in the normal manner by contacting your local representative or ibidi Customer Service at +49 (0)89/5204617 0. At its discretion, ibidi grants Not–For–Profit Entities a non–exclusive, personal, limited license to use this product for non–commercial life scienceresearch use only. Such license specifically excludes the right to sell or otherwise transfer this product, its components or derivatives thereof to thirdparties. No modifications to the protein coding sequence may be made without express written permission from ibidi. Any other use of this productrequires a license from ibidi. For–Profit Entities wishing to use this product are required to obtain a license from ibidi. For license information, pleasecontact a licensing representative by email at [email protected].
Salk Institute WPRE technology
Sirion has a limited license under patents owned by the Salk Institute for Biological Studies that permits Sirion to sell products containing WPRE forresearch use only, and not for any commercial uses. Excluded commercial uses include without limitation manufacturing, therapeutic, diagnostic andprophylactic uses, and any other commercial uses. use of this product by a purchaser for any purpose other than for research is unauthorized andprohibited. Administration to humans and use in human diagnostics is also prohibited. The Salk Institute actively licenses its patents for commercial uses,and a commercial use license may be available for Salk’s WPRE patents. If you wish to inquire about such a license, please contact: Office of TechnologyDevelopment, The Salk Institute for Biological Studies, 10010 North Torrey Pines Road, La Jolla, CA 92037, Phone: (858) 453-4100 extension 1278, Fax:(858) 546-8093.
Institut Pasteur cPPT technology
Limited license related to product code : 60141, 60142 This product and its use are the subject of one or more of patents: WO199/055892, FR9805197,AU2003/271326, AU2007/216712, CA 2326719, EP 1071804, EP 2292778, JP 4571306, US 6,682,907, US 7,981,671, US 8,367,068, and WO1999/055892,AU784910, AU2006/233199, CA 2382832, CN 1402793, EP 1224314, EP1792997, HK 1050210, IL 148900, JP 4436024, US 8,093,042, and/or other pendingforeign patent applications licensed to Sirion. The purchase of this product conveys to the buyer the non-transferable right to use the purchased amountof the product and components of the product in research conducted by the buyer (whether the buyer is an academic or for-profit entity). The buyercannot disclose information, sell or otherwise transfer this product, its components or materials made using this product or its components to a third partyor otherwise use this product or its components or materials made using this product or its components for any commercial purposes. If the buyer isnot willing to accept the limitations of this limited use statement, Sirion is willing to accept return of the product with a full refund. For information onpurchasing a license to the DNA-Flap technology for purposes other than research, contact the Transfer of Technology Office, Institut Pasteur, 28 rue duDocteur Roux, 75 724 Paris Cedex 15 (www.pasteur.fr).
Oxford BioMedica LentiVector® technology
This product is licensed under U.S. Pat. Nos. 5,817,491; 5,591,624; 5,716,832; 6,312,682; 6,669,936; 6,235,522; 6,924,123 and foreign equivalents from OxfordBioMedica (UK) Ltd., Oxford, UK, and is provided for non-commercial research purposes. To enhance basic understanding of biological mechanismsunderlying human and animal development, diseases and conditions, and for preclinical drug discovery and development including screening, andtarget validation but expressly excluding use in humans for therapeutic and for diagnostic purpose. Information about licenses for commercial usesexcluded under this license is available from Sigma-Aldrich Co. LLC, a Delaware limited liability company with a principal place of business at 3050Spruce Street, St. Louis, MO 63103. LentiVector is a registered US and European Community trade mark of Oxford BioMedica plc.
