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RheoSwitch® Cell Line CHO-R5 |
 | | | | RheoSwitch® Cell Line CHO-R5 | | | |
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Prices are in US dollars and valid only for US orders.
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Description:
The RheoSwitch® Cell Line CHO-R5 is part of the RheoSwitch® Mammalian Expression System. It is derived from Chinese Hamster Ovary CHO-K1 cells stably transfected with pNEBR-R1 Regulator Plasmid 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.
Supplied frozen in: Ham's F12 medium (F12K) containing 50% Fetal Bovine Serum and 10% DMSO.
Growth Medium and Conditions: Ham's F12 medium (F12K) with 1.5 g/L sodium bicarbonate, and 2 mM L-glutamine, 5% or 10% FBS, antibiotic-antimycotic (50 units/ml Penicillin G, 0.25 µg/ml Amphotericin B, & 50 µg/ml Streptomycin) and 500 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 Line CHO-R5. Cells were transfected in a 24-well plate with pNEBR-X1GLuc Control Plasmid (0.4 µg/well) using TransPass D2 (NEB #M2553) as described in the RheoSwitch manual. Quadruplicate 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 CHO-R5 was transfected with pNEBR-X1Hygro encoding a HA-tagged protein using TransPass D2 Transfection Reagent (NEB #M2554) according to manufacturer's intructions. RSL1 was added 16 hours later and cells harvested 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. If the frozen culture must be stored, the cells should be placed in liquid nitrogen vapor phase.
Usage notes:- Additional Materials Required: Growth Medium (see Growth Medium and Conditions below), 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 > 90% viability upon thawing. 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
pNEBR-X1GLuc Control Plasmid
pNEBR-X1Hygro Vector
RheoSwitch® Ligand RSL1
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).
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