Manual - New England Biolabs

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To induce expression, RSL1 ligand is added to the transfected cells. RSL1 ligand can
induce gene expression over a broad range of concentrations (Figure 3). Use a final
concentration of 500 nM for full induction. Cells can be assayed for expression 24
hours post induction. Maximum fold induction depends on cell type and transfection
efficiency. See the "Example Protocol" for transient transfection and induction in
NIH 3T3 cells.
3. Establish a stable cell line expressing the RheoSwitch Receptor:
The neomycin resistance gene in pNEBR-R1 can be used to construct stable cell lines
expressing the RheoReceptor and RheoActivator proteins. After transfection, cells
are selected via their ability to grow in media containing the antibiotic G418 using
standard techniques. G418-resistant clones can be screened for RheoSwitch function
using transient transfection of pNEBR-X1GLuc. Screen multiple clones and select
those with high RSL1 induction ratios. For a detailed protocol see:
Current Protocols in Molecular Biology, Chapter 16, Protein Expression, Section III,
Expression in Mammalian cells.
4. Introduce the cloned gene into the RheoSwitch Receptor stable cell line:
The cloned gene in pNEBR-X1Hygro can be introduced by transient transfection in
the stable cell line expressing the RheoSwitch Receptor and induced as described in
step 2. The hygromycin resistance gene in pNEBR-X1Hygro can be used to construct
a stable line expressing the cloned gene. After transfection, cells are selected via their
ability to grow on media containing hygromycin using standard techniques and assayed
for expression of the cloned gene.