CellSensor Lentiviral Vectors User Guide

Transcription

1 CellSensor Lentiviral Vectors User Guide Cat. nos. K1109, K1110, K1115, K1116, K1117, K1118, K1119, K1121, K1124, K1125, K1126, K1128, K1135, K1132, K1133, K1164, K1193, K1194, K1195, K1196, and K1242 Lit. No. O13404.pps Rev Date: 31 May 2006 Shipping Condition: Dry Ice Storage: 20 C TABLE OF CONTENTS 1.0 MATERIALS SUPPLIED MATERIALS REQUIRED, BUT NOT SUPPLIED VIRAPOWER LENTIVIRAL EXPRESSION SYSTEM OVERVIEW Description How the ViraPower Lentiviral Expression System Works LIPOFECTAMINE 2000 TRANSFECTION REAGENT Description How Lipofectamine 2000 Transfection Reagent Works ENHANCER ELEMENTS AVAILABLE BIOSAFETY LEVEL AND HANDLING PURCHASER NOTIFICATION MATERIALS SUPPLIED Description: CellSensor Lentiviral Vectors Shipping Condition: Dry Ice Storage Condition: 20 C Quantity: 10 µg 2.0 MATERIALS REQUIRED, BUT NOT SUPPLIED Reagents Recommended Source Cat. no. ViraPower Bsd Lentiviral Support Kit Invitrogen K Lipofectamine 2000 Transfection Reagent Invitrogen For Technical Support for this or any other Invitrogen Drug Discovery Solutions Products, dial , extension 40266

2 Invitrogen CellSensor Lentiviral Vectors User Guide O13404pps 09/05 Page 2 of VIRAPOWER LENTIVIRAL EXPRESSION SYSTEM OVERVIEW 3.1 Description CellSensor Lentiviral Vectors may be used in combination with the ViraPower Lentiviral Expression System for viral transduction of mammalian cells or analogous to a standard vector for transfection of mammalian cells with traditional lipid-based transfection techniques. Many mammalian cell types and experimental situations create challenges for standard transfection experiments. The ViraPower Lentiviral Expression System overcomes these challenges with efficient viral delivery to both dividing and non-dividing cells. Lentivirus is ideal for analysis of long-term gene expression, even in terminally differentiated or growth-arrested cells. Use this system to analyze gene expression beyond the limitations of traditional transfection reagents or other viral systems. The ViraPower Lentiviral Expression System provides the high levels of stable gene expression necessary for valid results in virtually any cell line. 3.2 How the ViraPower Lentiviral Expression System Works High-level stable gene expression is easily achieved with the ViraPower Lentiviral Expression system (Figure 1). Co-transfect the vector containing your gene of interest along with the optimized ViraPower Lentiviral Packaging Mix into 293FT producer cells. The ViraPower Packaging Mix provides a pre-optimized mix of plasmids that produce the lentiviral packaging proteins (gag/pol and rev) and the VSV-G envelope protein. The VSV-G envelope gives the particles produced the ability to transduce a broad spectrum of mammalian cells. In hours harvest the supernatant, which now has replication-incompetent viral particles that contain your gene of interest. Since the packaging proteins are provided in trans, these plasmids are not part of the final viral particles. You can use this supernatant to efficiently transduce any mammalian cell. Each transfection produces sufficient supernatant for multiple transduction experiments. Detailed protocols, citations, and additional product information for the ViraPower Lentiviral Expression System can be found at

3 Invitrogen CellSensor Lentiviral Vectors User Guide O13404pps 09/05 Page 3 of LIPOFECTAMINE 2000 TRANSFECTION REAGENT OVERVIEW 4.1 Description Lipofectamine 2000 Reagent is a cationic lipid that offers the highest transfection efficiencies of the widest variety of cell lines and high protein expression levels or gene inhibition (RNAi). It is the leading transfection reagent for simple, effective, and optimal nucleic acid delivery. Lipofectamine 2000 Reagent provides all of the following: Exceptional transfection efficiency in a broad range of cell lines and high levels of recombinant protein expression Superior performance for sirna delivery Proven efficacy in the presence of serum eliminates the need to change media following transfection A reagent proven to work in high-throughput applications A reliable reagent for establishing stable cell lines 4.2 How Lipofectamine 2000 Transfection Reagent Works Lipofectamine 2000 Reagent offers high transfection efficiencies for a broad range of cell types, including adherent and suspension cell lines. This means you can choose the most appropriate cell line for gene function studies or target validation. For cell lines commonly used for protein expression and cell-based functional assays, including COS-7, CHO and HEK 293T, efficiencies are often greater than 90%. Lipofectamine 2000 Reagent works effectively with many primary cell lines and other difficult to transfect cell lines. In addition to its versatility with diverse cell lines, Lipofectamine 2000 provides high transfection efficiencies using media with or without serum. For cells sensitive to the absence of serum, add DNA:lipid complexes directly to cells in serum-containing medium. For gene silencing, Lipofectamine 2000 Reagent s high efficiency transfections leads to the high levels of gene knockdown needed to achieve successful results. Simply mix Lipofectamine 2000 Reagent with nucleic acid, add to cell culture, and express protein or knockdown genes. The simplicity and speed combined with high transfection efficiency make Lipofectamine 2000 Reagent ideal for transient protein expression or high throughput RNAi. Transfection conditions can be easily established for automated or robotic systems used in such applications. Protocols, citations, and additional product information for Lipofectamine 2000 can be found in the Cell Lines Database at

4 Invitrogen CellSensor Lentiviral Vectors User Guide O13404pps 09/05 Page 4 of ENHANCER ELEMENTS AVAILABLE Cat. no. Product Element K1109 K1115 plenti-bsd/nfat-bla Vector plenti-bsd/isre-bla Vector Nuclear Factor of Activated T-Cells (NFAT) Response Interferon Stimulated Response Element (ISRE) Transcription Factor NFAT STAT1/STAT2 K1116 plenti-bsd/sie-bla Vector SIE STAT3 K1117 plenti-bsd/sbe-bla Vector Smad Binding Element (SBE) Smads K1118 plenti-bsd/ap-1-bla Vector Activator Protein 1 (AP-1) Response Element K1119 plenti-bsd/c-fos-bla Vector C-fos Promoter (SIE SRE CRE) c-jun/c-fos STAT3, ATF2/CREB, Pathway PKC and Ca 2+ /Calcineurin Proliferation/Inflammation Proliferation/Inflammation Transforming Growth Factor Beta (TGF-β) JNK JNK/p38/MAPK and K1110 plenti-bsd/cre-bla Vector camp Response Element (CRE) ATF2/CREB JNK/P38 and PKA K1121 plenti-bsd/sre-bla Vector Serum Response Element (SRE) Elk-1/SRF MAPK/JNK K1124 plenti-bsd/hre-bla Vector Hypoxia Responsive Elements (HRE) HIF Angiogenesis K1125 plenti-bsd/myc-bla Vector E Box DNA Binding Element Myc/Max Cell Proliferation K1126 plenti-bsd/lef/tcf-bla Vector Lymphoid Enhancing Factor (LEF)/T Cell Factor (TCF) DNA TCF/LEF Wnt-Frizzled K1128 plenti-bsd/nfκb-bla Vector Nuclear Factor of B (NFκB) Cells Response Element NFκB NFκB K1135 plenti-bsd/gli-bla Vector Gli Hedgehog Hedgehog K1164 plenti-bsd/gas-bla Vector Gamma-Interferon Activation Sequence (GAS) STAT1 Proliferation/Inflammation K1193 plenti-bsd/p53re-bla Vector p53 Response Element p53 Cell Growth/Apoptosis K1194 plenti-bsd/fhre-bla Vector Forkhead Response Element FOXO3 PI3K/Akt/Forkhead K1195 plenti-bsd/c.ebp-bla Vector C/EBP Response Element C/EBPα C/EBP (differentiation) K1196 plenti-bsd/dhfr(e2f) Vector E2F/DP1 Response Element E2F/DP1 RB-E2F/cell cycle K1242 plenti-bsd/stat6-bla Vector STAT6 STAT6 JAK/STAT (IL-4) 6.0 BIOSAFETY LEVEL AND HANDLING The plenti vectors should be handled at Biosafety Level 1 and virus produced by this method should be handled at Biosafety Level 2. If the vector is used analogous to a standard vector, all material may be used at Biosafety Level 1.

5 Invitrogen CellSensor Lentiviral Vectors User Guide O13404pps 09/05 Page 5 of PURCHASER NOTIFICATION Limited Use Label License No. 5: Invitrogen Technology The purchase of this product conveys to the buyer the non-transferable right to use the purchased amount of the product and components of the product in research conducted by the buyer (whether the buyer is an academic or for-profit entity). The buyer cannot sell or otherwise transfer (a) this product (b) its components or (c) materials made using this product or its components to a third party or otherwise use this product or its components or materials made using this product or its components for Commercial Purposes. The buyer may transfer information or materials made through the use of this product to a scientific collaborator, provided that such transfer is not for any Commercial Purpose, and that such collaborator agrees in writing (a) not to transfer such materials to any third party, and (b) to use such transferred materials and/or information solely for research and not for Commercial Purposes. Commercial Purposes means any activity by a party for consideration and may include, but is not limited to: (1) use of the product or its components in manufacturing; (2) use of the product or its components to provide a service, information, or data; (3) use of the product or its components for therapeutic, diagnostic or prophylactic purposes; or (4) resale of the product or its components, whether or not such product or its components are resold for use in research. Invitrogen Corporation will not assert a claim against the buyer of infringement of patents owned or controlled by Invitrogen Corporation which cover this product based upon the manufacture, use or sale of a therapeutic, clinical diagnostic, vaccine or prophylactic product developed in research by the buyer in which this product or its components was employed, provided that neither this product nor any of its components was used in the manufacture of such product. If the purchaser is not willing to accept the limitations of this limited use statement, Invitrogen is willing to accept return of the product with a full refund. For information on purchasing a license to this product for purposes other than research, contact Licensing Department, Invitrogen Corporation, 1600 Faraday Avenue, Carlsbad, California Phone (760) Fax (760) outlicensing@invitrogen.com Limited Use Label License No: 51 Blasticidin and the Blasticidin Selection Marker Blasticidin and the blasticidin resistance gene (bsd) are the subject of U.S. Patent No. 5,527,701sold under patent license for research purposes only. Inquiries for commercial use should be directed to: Kaken Pharmaceutical Company, Ltd., Bunkyo Green Court, Center Office Building, Fl, 28-8 Honkomagome 2-chome, Bunkyo-ku, Tokyo , Japan, Tel: ; Fax: Limited Use Label License No: 108 Lentiviral Technology The Lentiviral Technology (based upon the lentikat system) is licensed from Cell Genesys, Inc., under U.S. Patent Nos. 5,686,279; 5,834,256; 5,858,740; 5,994,136; 6,013,516; 6,051,427; 6,165,782 and 6,218,187 and corresponding patents and applications in other countries for internal research purposes only. Use of this technology for gene therapy applications or bioprocessing other than for non-human research use requires a license from Cell Genesys (Cell Genesys, Inc. 342 Lakeside Drive, Foster City, California 94404). The purchase of this product conveys to the buyer the non-transferable right to use the purchased amount of the product and components of the product in research conducted by the buyer, including non-gene therapy research and target validation applications in laboratory animals (whether the buyer is an academic or for-profit entity). The buyer cannot sell or otherwise transfer (a) this product (b) its components or (c) materials made using this product or its components to a third party or otherwise use this product or its components or materials made using this product or its components for Commercial Purposes. The buyer may transfer information or materials made through the use of this product to a scientific collaborator, provided that such transfer is not for any Commercial Purpose, and that such collaborator agrees in writing (a) not to transfer such materials to any third party, and (b) to use such transferred materials and/or information solely for research and not for Commercial Purposes. Commercial Purposes means any activity by a party for consideration and may include, but is not limited to: (1) use of the product or its components in manufacturing; (2) use of the product or its components to provide a service, information, or data; (3) use of the product or its components for therapeutic, diagnostic or prophylactic purposes; or (4) resale of the product or its components, whether or not such product or its components are resold for use in research. Invitrogen Corporation will not assert a claim against the buyer of infringement of the above patents based upon the manufacture, use or sale of a therapeutic, clinical diagnostic, vaccine or prophylactic product developed in research by the buyer in which this product or its components was employed, provided that neither this product nor any of its components was used in the manufacture of such product. If the purchaser is not willing to accept the limitations of this limited use statement, Invitrogen is willing to accept return of the product with a full refund. For information on purchasing a license to this product for purposes other than research, contact Licensing Department, Invitrogen Corporation, 1600 Faraday Avenue, Carlsbad, California Phone (760) Fax (760)

6 Invitrogen CellSensor Lentiviral Vectors User Guide O13404pps 09/05 Page 6 of 6 Limited Use Label License No. 150: GeneBLAzer Technology This product and/or its use is the subject of one or more of U.S. Patent Nos. 5,741,657, 5,955,604, 6,291,162, and 6,472,205 and foreign equivalents licensed to Invitrogen Corporation. The right to use this product for internal research specifically excludes the right to use this product to identify, discover, and profile compounds that act as a flavor, fragrance or taste-enhancers and modify a target identified in taste, olfaction, or pheromone detection, which compound does not require FDA approval of an NDA for claims of safety and efficacy. The right to use methods claimed in the foregoing patents with this product for research purposes can only be acquired by the use of GeneBLAzer substrates purchased from Invitrogen Corporation or its authorized distributors GeneBLAzer and CellSensor is a registered trademark of Invitrogen Corporation. Lipofectamine, and ViraPower are trademarks of Invitrogen Corporation Invitrogen Corporation. All rights reserved. For research use only. Not intended for any animal or human therapeutic or diagnostic use.