Manual - New England Biolabs

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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.
O
O
N
H
N
Figure 1: Chemical structure of RheoSwitch
Ligand RSL1 (MW=382.5 daltons).
O