 | | | | RheoSwitch® Mammalian Inducible Expression System |
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Prices are in US dollars and valid only for US orders.
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- Precise regulation of expression levels
- Negligible basal expression
- Synthetic inducer and engineered receptor eliminate non-specific side effects
- Greater than 1,000 fold induction levels routinely obtained
- No special culture and media requirements
Description:
The RheoSwitch® Mammalian Inducible Expression System permits maximum control of gene expression in mammalian cells. Analogous to the operation of a rheostat, the RheoSwitch technology allows induction and adjustable control of gene expression. This precise regulation of gene expression is achieved through the highly specific interaction of a synthetic inducer, RheoSwitch Ligand RSL1, and a chimeric bipartite nuclear receptor. This receptor is activated in the presence of RSL1 ligand, and the level of gene expression can be regulated by adjusting the concentration of RSL1 ligand in the tissue culture media.
The RheoSwitch technology offers several advantages over other inducible systems. Its precise control is unrivaled among mammalian expression systems, giving negligible levels of basal expression in the absence of inducer and greater than 10,000 fold induction when RSL1 ligand is present. Unlike other systems, which rely on steroids or other drugs, the synthetic ligand RSL1 shows no pleiotropic effects in mammalian cells and exhibits no cross talk with endogenous transcription factors. In addition, the RheoSwitch System has no special culture media requirements.
Figure 1: Chemical structure of RheoSwitch Ligand RSL1 (MW=382.5 daltons)
Figure 2: Transcriptional control using the RheoSwitch System. "OFF" state: In the absence of RSL1 ligand, the RheoReceptor and RheoActivator proteins exist in an inactive conformation and transcription is kept off. "ON" State: In the presence of RSL1 ligand, the two proteins stably dimerize and bind to the response element of pNEBR-X1Hygro in an active conformation and transcription is turned on.
Figure 3: Luciferase expression in response to increasing RSL1 concentrations. NIH 3T3 cells co-transfected with pNEBR-R1 and pNEBR-X1 expressing firefly luciferase were induced with the indicated RSL1 concentrations. Expression was measured 48 hours post-induction.
Figure 4: NIH 3T3 (A) and RheoSwitch HEK293-A7 (B) (NEB #C2003) cells were transfected with pNEBR-X1Hygro encoding a HA-tagged protein using TransPass D2 (NEB #M2554). NIH 3T3 cells were also co-transfected with pNEBR-R1. RSL1 was added to transfected cells 16 hours later, and cells were 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. A longer exposure of the HEK293-A7 immunoblot is shown (C) to illustrate that no protein is detected in the absence of RSL1.
Figure 5: Induction time course. NIH 3T3 cells co-transfected with pNEBR-R1 and pNEBR-X1GLuc control plasmid were induced with RSL1. Gaussia luciferase activity was assayed between 1 and 24 hours post-induction.
Figure 6: Rapid ON:OFF switch response to two cycles of addition and removal of RSL1. NIH 3T3 cells were co-transfected with pNEBR-R1 and pNEBR-X1 expressing a destabilized firefly luciferase and induced with RSL1 followed by removal of RSL1 at 24 hours. A second cycle of addition/removal of RSL1 results in a new ON:OFF switch response. To remove RSL1 and turn off expression, cells were washed and RSL1-free media was added at the times indicated. The blue line represents induced expression. The red line represents uninduced expression.
Background:
The RheoSwitch System represents the next generation of inducible gene expression systems for mammalian cells. It is composed of an engineered nuclear receptor and a synthetic ligand inducer that is highly specific. The system is built on a two-hybrid switch format that activates transcription in the presence of inducer and represses gene expression in its absence (1,2,3 and unpublished observations). The synthetic receptor is composed of two proteins, RheoReceptor-1 and RheoActivator, that dimerize to make a holoreceptor. Both are expressed from strong constitutive promoters on plasmid pNEBR-R1. The RheoReceptor-1 protein is a highly engineered ligand-binding domain (LBD) of an insect EcR nuclear receptor fused to the yeast GAL4 DNA binding domain. The RheoActivator protein is an insect/mammalian RXR hybrid LBD fused to the viral activation domain VP16. The gene to be expressed is cloned into the pNEBR-X1 plasmid under control of five tandem repeats of the GAL4 response element (5XRE). In the absence of RSL1 ligand, the receptor represses transcription by binding to the GAL4 elements in a transcriptionally inactive conformation. Upon induction, the RSL1 ligand tightly binds and changes the conformation of the RheoReceptor-1 protein which stabilizes the holoreceptor heterodimer on the 5XRE. The activated holoreceptor and the VP16 activation domain bind and recruit to the promoter transcriptional coactivators along with basal machinery, resulting in a highly induced transcriptional state (Figure 2).
This high induction of transcription coupled with extremely low basal expression results in extremely high induction levels (greater than 10,000 fold induction), with a degree of control that is superior to other systems.
RSL1 ligand induces transcription over a broad range of concentrations from picomolar to micromolar. As illustrated in Figure 3, Gaussia Luciferase activity increases in an almost linear fashion with increasing amounts of RSL1. Likewise, increasing amounts of protein are observed, via immunoblot assay, with increasing amounts of RSL1 (Figure 4). Even with a longer exposure, no protein is observed in non-induced cells (Figure 4, Lane 5), further demonstrating the degree of control of over expression. Induced expression can be seen in as little as 1 hour, reaching a maximum level between 48 and 72 hours (Figure 5). The RheoSwitch System is very responsive to RSL1 and its removal from the medium will rapidly turn off gene expression (Figure 6).
RSL1 ligand is a synthetic diacylhydrazine, [N-(2-ethyl-3-methoxybenzoyl)-N´-(3,5-dimethylbenzoyl)-N´-tert-butylhydrazine] (Figure 1) (4). It is one of a family of compounds that have been found to act as non-steroidal ecdysone agonists and can function as gene inducers (4,5). RSL1 binds tightly and selectively to RheoReceptor-1, altering its conformation so that it releases bound negative regulatory cofactors and stabilizes the RheoReceptor/RheoActivator heterodimer. RSL1 is not a steroid,it is invisible to mammalian nuclear receptors and has been shown to be inert within all cell lines tested to date. The result is an inducible system that specifically controls the gene of interest without unwanted effects on the host cells.
Applications:- Inducible protein expression
- Expression of toxic genes
- Overexpression/mutant rescue studies
Kit Components:
Gaussia Luciferase Assay Kit
pNEBR-R1 Regulator Plasmid (500 μg/ml)
pNEBR-X1GLuc Control Plasmid (500 μg/ml)
pNEBR-X1Hygro Vector
RheoSwitch® Ligand RSL1
GLuc Assay Buffer
GLuc Substrate (100X)
RheoSwitch® R-X1 Sequencing Primer (17-mer) (200 pmol)
Storage Conditions
Storage Temperature:
-20°C
FAQs
- Where can I find more detailed FAQs for RheoSwitch® Mammalian Inducible Expression System?
- What cell lines and systems can be used with RheoSwitch?
- Can RheoSwitch be used in mice?
- Is RSL1 stable at room temperature?
- Can RSL1 be diluted in media?
- I have cloned my gene into pNEBR-X1. Can I make a stable cell line with this plasmid?
- How can I assay for expression of the RheoReceptor and RheoActivator genes?
- Can I make a stably transformed cell line using pNEBR-R1?
- What concentration of G418 should I use to construct a stable cell line with pNEBR-R1?
Figure 6A: pNEBR-X1Hygro plasmid map. Used to clone the gene of interest, it has five copies of the GAL4 response element (5XRE) upstream of a TATA box and a short leader sequence followed by multiple cloning sites and an SV40 polyA signal. Only unique restriction enzyme sites are shown.
Figure 6B: pNEBR-X1Hygro multiple cloning sites (MCS). To minimize uninduced expression, a short sequence containing the TATA box separates the end of the 5XRE from the predicted transcription start. Only unique restriction enzyme sites are shown.
Figure 7: pNEBR-R1 plasmid map. Expresses an engineered nuclear receptor heterodimer, consisting of two proteins, RheoReceptor-1, and RheoActivator. The two proteins constitute the holoreceptor and regulate transcription of genes cloned into the expression vector, pNEBR-X1. The two proteins are expressed from the constitutive UbC and UbB promoters, respectively. The plasmid also contains the neomycin resistance gene under control of the SV40 early promoter for the generation of stable cell lines.
References
- Palli, S.R., Kapitskaya, M.Z., Kumar, M.B. and Cress, D.E. (2003) Eur. J. Biochem., 270, 1308-1315.
- Dhadialla, T.S., Carlson, G.R. and Le, D.P. (1998) Annu. Rev. Entomol., 43, 545-569.
- Kumar, M.B., Potter, D.W., Hormann, R.E., Edwards, A., Tice, C.M., Smith, H.C., Dipietro, M.A., Polley, M., Lawless, M., Wolohan, P.R., Kethidi, D.R. and Palli, S.R. (2004) J. Biol. Chem., 279, 27211-27218.
- Verhaegent, M. and Christopoulos, T.K. (2002) Anal. Chem., 74, 4378-4385.
Reagents Sold Separately
Gaussia Luciferase Assay Kit
pNEBR-R1 Regulator Plasmid
pNEBR-X1GLuc Control Plasmid
pNEBR-X1Hygro Vector
RheoSwitch® Ligand RSL1
RheoSwitch® R-X1 Sequencing Primer (17-mer)
Companion Products
pNEBR-X1 Vector
RheoSwitch® Cell Line CHO-R5
RheoSwitch® Cell Line CHO-R5 and pNEBR-X1GLuc Control Plasmid
RheoSwitch® Cell Line HEK293-A7
RheoSwitch® Cell Line HEK293-A7 and pNEBR-X1GLuc Control Plasmid
RheoSwitch® Cell Line NIH3T3-47
RheoSwitch® Cell Line NIH3T3-47 and pNEBR-X1GLuc Control Plasmid
TransPass™ D1 Transfection Reagent
TransPass™ D2 Transfection Reagent
Legal
Licenses/Patents/Disclaimers:
RheoSwitch® Mammalian Inducible Expression System Notice to Purchaser: This product is covered by patents owned or licensed by RheoGene, Inc. and licensed to New England Biolabs, Inc. 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 converys 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 obtain a commercial research license from RheoGene, Inc. in order to continue use of the RheoSwitch® System.
pNEBR-X1-GLuc Gaussia Luciferase Control Plasmid 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.
pNEBR-X1GLuc Gaussia Luciferase Control Plasmid 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 entties; AND ADDITIONALLY agree to an are hereby bound to use the materials FOR EVALUATION PURPOSES ONLY. Commercial Recipient hereby agrees to destroy and cease use of any materials or derivatives containing any portion of these materials within 120 days from receipt. Commercial Recipient agrees not to use the materials for any use, other than the 120 day suitability evaluation without prior written permission or obtaining a valid license from Prolume Ltd./Nanolight Technologies
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