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RheoSwitch® Cell Line HEK293-A7 and pNEBR-X1GLuc Control Plasmid |
 | | | | RheoSwitch® Cell Line HEK293-A7 and pNEBR-X1GLuc Control Plasmid | | | |
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Prices are in US dollars and valid only for US orders.
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Description:
The RheoSwitch® Cell Line HEK293-A7 is part of the RheoSwitch® Mammalian Expression System. It is derived from human HEK 293 cells stably transfected with pNEBR-R1 Vector and selected for optimal RSL1 inducible expression properties. The cells can be used to express a gene of interest in transient transfections using pNEBR-X1 Vector (NEB #N8078). Alternatively, a stable cell line expressing both the RheoSwitch Receptor and the gene of interest can be constructed using pNEBR-X1Hygro Vector (NEB #N8083) and selecting for both neomycin and hygromycin resistance. The plasmid pNEBR-X1GLuc Control Plasmid (NEB #N8080) is included to be used as a positive control for HEK293-A7 in transient transfection experiments.
Supplied frozen in: DMEM (high glucose) containing 50% Fetal Bovine Serum and 10% DMSO.
Growth Medium and Conditions: DMEM (high glucose, 3.7 g/l sodium bicarbonate, 2 mM L-glutamine) containing 5% or 10% FBS, antibiotic-antimycotic (50 units/ml Penicillin G, 0.25 µg/ml Amphotericin B, & 50 µg/ml Streptomycin) and 300 µg/ml G418. Cultures should be grown with 5% CO2 in atmosphere at 37°C.
Figure 1: RSL1 induction of GLuc expression in Rheoswitch Cell Liine HEK293-A7. Cells were transfected in a 24-well plate with pNEBR-X1GLuc Control Plasmid (0.5 µg/well) or the constitutively expressed pCMV-GLuc (1 µg/well) using TransPass D2 (NEB #M2553) as described in the RheoSwitch manual. Triplicate wells were treated with DMSO or 500 nM RSL1 overnight. 20 µl of the culture supernatants were assayed with the Gaussia Luciferase Assay Kit (NEB #E3300).
Figure 2: Immunoblot Assay of RSL1 Induction. RheoSwitch Cell Line HEK293-A7 was transfected with pNEBR-X1Hygro encoding a HA-tagged protein using TransPass D2 Transfection Reagent (NEB #M2554) according to manufacturer's instructions. RSL1 was added 16 hours later and cells harvest 24 hours after induction (Note: DMSO is added as a control for 0 nM RSL1). Harvested cells were lysed and equivalent amounts of protein (as determined by Coomassie Stain) were electrophoresed and transferred to nitrocellulose. An immunoblot assay was performed on the filter using anti-HA as primary antibody and detected using anti-mouse secondary antibody and reagents.
Reaction & Storage Conditions
Storage Temperature:
-196°C
Store in Liquid Nitrogen
Notes
General notes:- Storage: To ensure the highest level of cell viability, it is recommended that the frozen cell vial be thawed and the culture initiated immediately upon arrival. The frozen cells may be stored temporarily (for 24 hours) at -70°C. Cells previously stored at -70°C can not be refrozen in liquid nitrogen vapor phase.
Usage notes:- Additional Materials Required: Growth Medium (see Growth Medium and Conditions section), sterile 0.25% Trypsin solution, Cryoprotectant Medium (see Protocol for preparation of frozen stock), and isopropanol-filled cryo 1°C freezing container.
Quality Control for Current Lot
Quality control values for a specific lot can be found on the datacard which accompanies each product.
Quality Assurance Statement:
Cells are mycoplasma free and have. The cell density should be approximately 30-40% confluent 48 hours after the initial revival of the frozen cells. Cells show strong RSL1-dependent expression of an RSL1-inducible plasmid (see Figure 1).
References
- Freshney, R. I. (1994) A manual of Basic Technique (3rd ed.), New York: Alan R. Liss.
- RheoSwitch® Mammalian Inducible Expression Manual, unpublished observations.
Companion Products
pNEBR-X1 Vector
RheoSwitch® Ligand RSL1
RheoSwitch® Mammalian Inducible Expression System
TransPass™ D1 Transfection Reagent
TransPass™ D2 Transfection Reagent
Legal
Licenses/Patents/Disclaimers:
This product is the subject of one or more of U.S. Patent Nos. 5,514,578, 6,245,531, including the corresponding foreign patents (Australian patent No. 752699, Canadian Patent Application No. 2076386, European Patent No. 0517805) and patent applications, U.S. Patent Nos. 6,258,603, 5,503,028 and U.S. Patent Applications 09/965,703, 10/239,134, 10/468,199, 10/468,193, licensed to New England Biolabs (NEB).
The purchase of this product conveys to the non-commercial purchaser the non-transferable right to use the purchased amount of the product and components of the product for research use only conducted by the purchaser. For commercial purchasers using this product for research, the purchase of this product conveys to the purchaser the non-transferable right to use the purchased amount of the product and components of the product for a six (6) month evaluation period after the date of purchase for research use only. After the six (6) month evaluation period, the purchaser must contact RheoGene, Inc. to obtain a commercial research license.
The purchaser 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 purchaser cannot use this product or its components in whole plants, plant cells or humans. The purchaser cannot directly or indirectly disassemble, decompile, translate, reverse engineer, modify or adapt, (a) this product or (b) its components. The purchaser may transfer information or materials made through the use of this product to a scientific collaborator, provided that such transfer is not for the commercial purposes of the purchaser, 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 products or its components for therapeutic, diagnostic or prophylactic purposes; (4) use of the product or its components to screen for agonists to the RheoSwitch® System; or (5) resale of the product or its components, whether or not such product or its components are resold for use in research. If the purchaser is not willing to accept the limitations of the limited use statement, NEB is willing to accept return of the products with a full refund. For information on commercial use of this product or obtaining a license to this product for purposes other than research, contact RheoGene, Inc. by calling Dr. Camille Jolly-Tornetta, Director of Intellectual Property (610-650-8734, ext. 104).
Statement for pNEBR-X1Gluc. By opening this package or by using the materials enclosed within: Recipient is legally bound and accepts the following terms and conditions: Non-Commercial entities: This product is covered by US Patents # 6,232,107 and other patents that are the legal property as assigned to Prolume Ltd./Nanolight Technologies. This product is licensed only to the purchasing laboratory-research group. Recipient agrees not to transfer this plasmid or derivatives of this vector to any other laboratory, person or research group, even if within the same institution.
Recipient agrees not to alter or make any changes to the nucleotide coding sequence or secretory coding sequence of the luciferase(s) contained within without prior written permission from Prolume Ltd./Nanolight Technologies (www.nanolight.com). Recipient agrees not to file for any patent rights or to any inventions claiming any portion of the luciferase(s) within this material without prior written permission from Prolume Ltd./Nanolight Technologies.
Commercial For-Profit Entities & Non-Profit Foundations (herein referred to as Commercial Recipients): Commercial Recipients wishing to derive products, engage in the sale or license of any products, discover drugs, or make inventions by use of the materials enclosed, fully agree to the terms mentioned above for Non-Commercial entities; AND ADDITIONALLY agree to and are hereby bound to use the materials FOR EVALUATION PERFORMANCES ONLY. Commercial Recipient hereby agrees to destroy and cease use of any materials or derivatives containing any portion of these materials within 180 days from receipt. Commercial Recipient agrees not to use the materials for any use, other than the 180 day suitability evaluation without prior written permission or obtaining a valid license from Prolume Ltd./Nanolight Technologies.
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