Manual - New England Biolabs

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4 Introduction: 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. Applications: • Inducible protein expression • Expression of toxic genes • Overexpression/mutant rescue studies Features: • Precise regulation of expression levels • Negligible basal expression • Synthetic inducer and engineered receptor eliminate non-specific side effects
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format that activates transcription in the presence of inducer and represses gene expression in its absence (1,2,3 and unpublished observations).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 5
Page 1: Catalog #E3000S Store at -20°C Rhe
Page 4 and 5: 2 Supplied Components: The RheoSwit
Page 8 and 9: 6 amounts of RSL1 (Figure 4). Even
Page 10 and 11: 8 Luciferase Activity (RLU) 90,000
Page 12 and 13: Luciferase Activity (RLU) 10 100,00
Page 14 and 15: 12 Gaussia Luciferase (GLuc) Contro
Page 16 and 17: 14 To induce expression, RSL1 ligan
Page 18 and 19: 16 4. Add RSL1 ligand to a final co
Page 20 and 21: 18 B. PshA I 3640 BfuA I - BspM I 3
Page 22 and 23: 20 References: 1. Karzenowski, D.,
Page 24: 22 Licensing Information: RheoSwitc

Manual - New England Biolabs

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