Manual - New England Biolabs

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12 Gaussia Luciferase (GLuc) Control Reporter The RheoSwitch Mammalian Inducible Expression System includes a control reporter plasmid (pNEBR-X1GLuc) encoding a secreted luciferase. GLuc is a new luciferase from the marine copepod Gaussia princeps (6). This luciferase, which does not require ATP, catalyzes the oxidation of the substrate coelenterazine in a reaction that produces light (470 nm), and has considerable advantages over other luminescent reporter genes. Features of GLuc Control Reporter: • It possesses a natural secretory signal and upon expression is secreted in the cell culture medium facilitating assays. • Produces 1,000 fold higher light intensity than Renilla or firefly luciferases. • Multiple samples can be assayed from the same transfected cells, for example, at different time points. • The sequence has been “codon-optimized” for expression in mammalian cells. • The secreted protein is very stable and has extremely high activity in light production allowing for very sensitive assays.
Experimental Outline: Expressing your gene of interest with the RheoSwitch System is accomplished in the following steps: • Clone gene of interest into the pNEBR-X1Hygro Vector. • Perform transient transfection and test induction of cloned gene. • Establish a stable cell line expressing the RheoSwitch Receptor. • Introduce cloned gene into the RheoSwitch Receptor stable cell line. 1. Clone gene of interest into the pNEBR-X1Hygro Vector: Clone your gene of interest into pNEBR-X1Hygro using any restriction site in the multiple cloning site (MCS) (Figure 6). The gene sequence must have its own functional ATG initiation and termination (stop) codons. Sequence at the 5´ junction of the cloned gene can be confirmed using primer NEB #S1280 (included in the kit) and at the 3´ end with NEB #S1272. 2. Perform transient transfection and test induction of cloned gene: Once the gene of interest has been cloned, test its expression in the desired cell line by co-transfecting with the RheoSwitch Receptor expressing plasmid, pNEBR-R1. Culture conditions and transfection protocols will depend on the particular cell line used. We recommend our TransPass D1 (NEB #M2553) or TransPass D2 (NEB #M2554) transfection reagents; see for protocols and additional information. A molar ratio of receptor plasmid to expression plasmid between 1:2 and 1:8 is recommended. The supplied pNEBR-X1GLuc can be used as a positive control. 13
Page 1: Catalog #E3000S Store at -20°C Rhe
Page 4 and 5: 2 Supplied Components: The RheoSwit
Page 6 and 7: 4 Introduction: The RheoSwitch ® M
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 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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