Functional characterization of ecdysone receptor gene
switches in mammalian cells
Siva K. Panguluri
1
, Prasanna Kumar
2
and Subba R. Palli
1
1 Department of Entomology, College of Agriculture, University of Kentucky, Lexington, KY, USA
2 RheoGene Inc., Norristown, PA, USA
Inducible expression of transgene is desirable for var-
ious applications such as functional genomics, gene
therapy, therapeutic protein production and tissue
engineering [1–4]. Many laboratories have designed a
number of gene switches to achieve inducible expres-
sion. However, many of these systems have their own
advantages and disadvantages, such as differences in
background, levels of induction, as well as unpredicta-
bility of their performance in vivo, mainly because of
the inadequate understanding of molecular mecha-
nisms of their function in vivo.
An insect hormone receptor based gene regulatory
system, ecdysone receptor (EcR) gene switch, is gain-
ing popularity due to its low background activity in
the absence of ligand, and high inducible expression in
the presence of synthetic nonsteroid ligands that have
shown very good safety profiles (as reviewed in [1]).
The EcR is a member of the nuclear receptor super-
family [2] which is composed of N-terminal A ⁄ B
domain, the DNA binding C domain, the D domain
(hinge region), ligand binding E domain, and C-ter-

response elements in the absence of ligand. The VP16(AD) fusion protein
of a chimera between human and locust RXR could heterodimerize with
GAL4(DBD):EcR(LBD) in the absence of ligand but the VP16(AD) fusion
protein of Homo sapiens RXR requires ligand for its heterodimerization
with GAL4(DBD):EcR(LBD).
Abbreviations
AD, activation domain; Cf, Choristoneura fumiferana; ChIP, chromatin immunoprecipitation; DB, Drosophila ⁄ Bombyx; DBD, DNA binding
domain; Dm, Drosophila melanogaster; EcR, ecdysone receptor; GE, GAL4:CfEcR; GST, glutathione S-transferase; Hs, Homo sapiens; LBD,
ligand binding domain; Lm, Locusta migratoria; RE, response element; RXR, retinoid X receptor; USP, ultraspiracle.
5550 FEBS Journal 273 (2006) 5550–5563 ª 2006 The Authors Journal compilation ª 2006 FEBS
nuclear receptors [1], noticeably with ultraspiracle
(USP), an orthologue of the vertebrate retinoid X
receptor (RXR). EcR functions as a ligand controlled
transcription factor, which makes it a suitable constitu-
ent of gene switches. The main advantage of EcR gene
switches in mammalian system is that the ecdysteroids,
which are used to induce the switch, are structurally
different from mammalian steroids, and they are not
expected to bind to vertebrate steroid receptors. More-
over, humans eat large amounts of phytoecdysteroid-
containing vegetables without any detrimental effects
[3]. Many versions of EcR gene switches were devel-
oped in different laboratories to achieve high degree of
induction.
Initially, Christopherson et al. [4] transfected a
HEK293 cell line with Drosophila melanogaster EcR
(DmEcR) and a reporter gene to test various ecdyster-
oids and vertebrate steroids. Ponasterone A and Mur-
esterone A (MurA) were active and MurA could also
induce reporter gene when used with estrogen response

to the bipartite EcR gene switch. The current versions
of bipartite EcR gene switch have very low background
activity of reporter gene in the absence of ligand and
are activated by subnanomolar concentrations of lig-
and. One concern about this system for in vivo applica-
tions is the use of RXR, which is involved in large
number of metabolic pathways.
In the light of the potential use of this EcR gene
switch for in vivo applications such as gene therapy,
understanding the mechanism of functioning of this
switch in a mammalian system and improving the switch
to reduce any potential pleiotropic effects are therefore
necessary. The data presented here showed that the use
of two mutant versions of EcR (EcRtvy and EcRtvay)
increased ligand sensitivity of EcR gene switch. EcR
gene switch in a monopartite format containing a fusion
protein of VP16 activation domain, GAL4 DNA bind-
ing domain and the EcR DEF domains supported
ligand inducible reporter activity levels that are higher
than those supported by the other versions of EcR gene
switches reported so far. We also discovered that EcR
fusion proteins require heterodimeric partner for bind-
ing to ligand as well as for transactivation of reporter
genes in cells. An EcR gene switch that includes a fusion
protein containing VP16 activation domain, GAL4
DNA binding domain and EcR DEF domains and a
chimera containing helices 1–8 from human RXR
(HsRXR) and helices 9–12 from locust (Locusta migra-
toria; LmRXR) supported lower background levels of
reporter activity in the absence of ligand, higher indu-

switch showed the lowest induction levels and very low
basal activity. The VG(65)E(CDEF) format switch
showed highest fold induction (706-fold) at 25 lm con-
centration of ligand. Similar results were observed with
the GVE format switch (Fig. 2B) except with higher
background expression of reporter gene than that
observed for VGE format switch. The initial versions
of GEV format switches were made by replacing
helix 12 of EcR with VP16 activation domain. This
version of GEV supported constitutive expression of
reporter gene expression in the absence of ligand (data
not shown). We then modified GEV format switch to
include helix 12 of EcR and this version supported
ligand inducible reporter activity (see below).
To understand the variation in the performance of
monopartite switch in different cell lines, we tested the
VGE version in four different cell lines, HEK293,
A
B
Fig. 1. (A) Schematic diagram of constructs used in various experiments. (B) Chemical structures of ligands used in various experiments.
Functional characterization of EcR gene switches S. K. Panguluri et al.
5552 FEBS Journal 273 (2006) 5550–5563 ª 2006 The Authors Journal compilation ª 2006 FEBS
CHO, NIH 3T3 and CV1. Clear differences among
four cell lines in induction levels as well as fold
induction were observed (Fig. 3). The 3T3 cells showed
less basal expression in the absence of ligand and
therefore high fold induction when compared to the
other three cell lines. To determine if the difference in
the induction levels of reporter gene by EcR gene
switch is due to the differences in levels of HsRXR iso-

25
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0 0 0 5 1
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) F E D ( E V ) 7 4 1 ( G ) F
E
D
C (
E
V
) 7 4 1 ( G ) F
E D
( E V )
3 9
( G ) F E D C (
E
V ) 3 9 ( G ) F
E
D ( E V ) 5 6 (
G
) F E D
C
( E V ) 5 6 ( G
Normalised RLU

0.2
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25
Fig. 2. (A) Induction of luciferase reporter gene by monopartite switches. (A) Plasmid DNA samples of VP16(AD):GAL4(DBD)(1–147):EcR(C-
DEF) [VG(147)E(CDEF)] or VP16(AD):GAL4(DBD)(1–147):EcR(DEF) [VG(147)E(DEF)] or VP16(AD):GAL4(DBD)(1–93):EcR(CDEF) [VG(93)E(C-

(4316-fold) or wtEcR (4621-fold) at 5 lm RG-102240
(Fig. 5). Similar differences in the activity of EcRtvy,
EcRtvay and wtEcR gene switches were observed in
HEK293 cells (data not shown).
The monopartite gene switch format consists of the
DEF domains of mutant CfEcR (EcRtvy and
EcRtvay), GAL4 DNA binding domain and VP16
activation domain. Various combinations of the EcR
ligand binding domain, VP16 activation domain and
GAL4 DNA binding domain were constructed
(Fig. 1A) and tested in NIH ⁄ 3T3 cells. Out of six
combinations tested, VGEtvy (VP16:GAL4:EcRtvy)
showed the highest level of induction (1289-fold) in
cells treated with 5 lm RG-102240 (Fig. 6A). The
reporter gene induction was dose dependent and signi-
ficant levels of reporter gene induction were observed
even at lower concentration (0.2 lm) of RG-102240.
The VGEtvay (VP16:GAL4: EcRtvay) also showed
dose dependent induction with maximum induction of
53-fold in cells treated with 5 lm RG-102240 (Fig. 6B).
The low levels of induction in VGEtvay, when com-
pared to the VGEtvy, are not only due to low levels of
induced reporter activity but also due to high back-
ground activity in the absence of ligand. Although the
0
005
0001
0051
00
02

COHC
3
92
Normalised RLU
5
4
1
222
951
506
)3.8()5()8.21()3.9(
Fig. 3. NIH ⁄ 3T3 cells showed high induction levels of reporter
gene. The plasmid DNA samples of VP16:GAL4:EcR(DEF) [VGE]
and pFRLuc were transfected into 293, CHO, CV1 and 3T3 cells
using superfect lipid reagent. The transfected cells were grown in
the medium containing 0, 0.1, 1, 5, 10 and 50 l
M concentrations of
RG-102240. The cells were harvested at 48 h after adding ligand,
and the reporter activity was measured and the RLU presented are
mean ± SD (n ¼ 3). Numbers above the bars represents the maxi-
mum fold induction observed for that particular combination. The
numbers in parentheses represents the RLU of the reporter activity
in cells that exposed to dimethylsulfoxide (no ligand).
0
00001
00002
00003
00004
00005
00006

V
RcE:G
RXR:V
RcE:G
RXR:V
Fig. 4. The GAL4:EcR + VP16:RXRc switch showed higher induction
than the switches containing a and b isoforms of RXR. The plasmid
DNA samples of GAL4:EcR (GE) + VP16:HsRXRa ⁄ RXRb ⁄ RXRc and
pFRLuc were transfected into 3T3 cells using superfect lipid reagent.
The transfected cells were grown in the medium containing 0, 0.1, 1,
5, 10 and 50 l
M concentrations of RG-102240. The cells were har-
vested at 48 h after adding ligand. The reporter activity was meas-
ured and the RLU presented are mean ± SD (n ¼ 3). Numbers above
the bars represents the maximum fold induction observed for that
particular combination. The numbers in parentheses represents the
RLU of the reporter activity in cells exposed to dimethylsulfoxide (no
ligand).
0
0002
0004
0006
0008
000
0
1
00021
DMSO
0.2
1

grown in the medium containing 0, 0.2, 1 and 5 l
M concentrations
of RG-102240. The cells were harvested at 48 h after adding ligand
and the reporter activity was measured. The RLU presented are
mean ± SD (n ¼ 3). Numbers above the bars represents the maxi-
mum fold induction observed for that particular combination. The
numbers in the parenthesis represents the RLU of the reporter
activity in cells exposed to dimethylsulfoxide (no ligand). DMSO,
dimethylsulfoxide.
Functional characterization of EcR gene switches S. K. Panguluri et al.
5554 FEBS Journal 273 (2006) 5550–5563 ª 2006 The Authors Journal compilation ª 2006 FEBS
GVE combinations GAL4:VP16:EcRtvy (GVEtvy)
and GAL4:VP16:EcRtvay (GVEtvay) showed high
sensitivity to the ligand (showed induced reporter
activity even at low concentrations of 0.02 lm RG-
102240), they were leaky, showing high background
expression of reporter gene in the absence of ligand.
The GEV combinations GAL4:EcRtvy:VP16 (GEtvyV)
and GAL4:CfEcRtvay:VP16 (GEtvayV) showed low
background in the absence of ligand, but they also
showed very low levels of reporter induction in the
presence of ligand (19.6-fold and 9.7-fold, respectively,
at 5 lm RG-102240 concentration). Thus, out of the
three formats of monopartite switches tested, VGE for-
mat appeared to perform better than GVE or GEV.
Out of the two mutants tested, EcRtvy showed the
highest ligand sensitivity, low background and high
levels of ligand-induced reporter expression.
EcR fusion proteins need heterodimeric partner
for ligand binding

freedom of 10) and 1.447 nm (confidence interval of
0.9588–1.935, R2 of 0.9788, degree of freedom of 28),
respectively. These data showed that VGE fusion pro-
tein of EcR requires heterodimeric partner for binding
to the ligand.
Addition of RXR improves the performance
of monopartite gene switch
Because ligand binding assays showed that USP is
required for binding of ligand to EcR, we decided to
test whether RXR is required for efficient functioning
of VGEtvy switch. For this purpose we transfected
pFRLuc reporter, VGEtvy and one of the RXRs
(VHsRXR, VLmRXR or VP16 fusion of one of the
Hs-LmRXR chimeras) into NIH ⁄ 3T3 cells and the
transfected cells were exposed to various concentra-
tions of RG-102240. Chimeras 6, 8, 9 and VLmRXR
increased the performance of VGEtvy switch. Chimera
10, 11 and VHsRXR caused much lower enhancement
of VGEtvy switch when compared to VLmRXR or
VHs-LmRXR chimeras 6, 8, and 9 (Fig. 8A). Thus,
inclusion of VP16 fusion of one of the three chimeras
between HsRXR and LmRXR or LmRXR itself
caused substantial increase in the performance of
VGEtvy switch. Next, we wanted to determine whether
the presence of VP16 on both the receptors (VGEtvy
and VRXR) is responsible for the improvement in the
performance of VGEtvy switch in the presence of
VP16:RXR. VP16 domain was deleted from VHs-
0
2000

(3.7)
4917
1289
2.9
19.6
G:EcRtvay+
V:Hs-LmR
B
0
1000
2000
3000
4000
5000
6000
7000
8000
DMSO
0.2
1
5
DMSO
0.2
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5
DMSO
0.2
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DMSO

ability to improve the performance of VGEtvy and
GEtvyV in NIH ⁄ 3T3 cells. As shown in Fig. 8B,
Hs-LmRXR with and without VP16 fusion protein
performed equally well in improving the performance
of both VGEtvy and GEtvyV switches. Similar results
were observed when Hs-LmRXR chimera 9 with
and without VP16 was compared as a partner for
VGEtvay and GEtvayV (Fig. 8C). Thus, RXR region
but not the additional VP16 domain is responsible for
improving the performance of VGEtvy and GEtvyV
switches.
VGE fusion proteins utilize endogenous RXR for
efficient transgene activation
The ligand binding experiments showed that the ligand
binding to EcR improved in the presence of USP. The
transactivation experiments showed that the VGE
fusion protein can support the reporter gene induction
in the absence of exogenously supplied RXR but the
reporter activity was higher in the presence of exogen-
ously supplied RXR chimeras. It is then possible that
the endogenous RXRs in the cells are binding to VGE,
enabling them to transactivate genes placed under their
control. We tested this hypothesis using double-stran-
ded RNA prepared against HsRXRa and HsRXRb.
Microarray analysis of gene expression in HEK293
cells showed that only HsRXRa and HsRXRb are
expressed in these cells and HsRXRc expression is not
detected (data not shown). HEK293 cells were trans-
fected with VGEtvy and pFRLuc or VGEtvy and
pFRLuc and double-stranded RNA for RXRa and

M). The unbound ligand was
removed by dextran coated activated charcoal treatment and radioac-
tivity in the supernatant was counted using liquid scintillation coun-
ter. The specific binding was expressed in disintegrations per minute
and the error bars shown are the standard deviation of three repli-
cates. (B,C) Saturation kinetics of the binding of [
3
H]-labeled RH-2485
to mutant CfEcRtvy + CfUSP (B) or CfEcRtvy fusion protein in mono-
partite format (VGEtvy) plus CfUSP (C). Specific binding (d) which is
the total binding (j) minus the nonspecific binding (m) and dissoci-
ation constants (K
d
) are displayed.
Functional characterization of EcR gene switches S. K. Panguluri et al.
5556 FEBS Journal 273 (2006) 5550–5563 ª 2006 The Authors Journal compilation ª 2006 FEBS
VGEtvy+
V:HsR
A
VGEtvy+
V:Hs-LmR 6
VGEtvy+
V:Hs-LmR 8
VGEtvy+
V:Hs-LmR 9
VGEtvy+
V:Hs-LmR 10
VGEtvy+
V:Hs-LmR 11
VGEtvy+

0.008
0.04
0.2
DMSO
0.008
0.04
0.2
Normalised RLU
771
822
2131
7291
6.389
4141
)01()162()3.5()01()11()26()3.5(
B
VGEtvy VGEtvy+
V:Hs-LmR
VGEtvy+
Hs-LmR
GEtvyV+
V:Hs-LmR
GEtvyV+
Hs-LmR
0
0005
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2
41
9821
)4.11()5.6()3
(
)6
.
3(
)
2.8
(
C
VGEtvay VGEtvay+
V:Hs-LmR
VGEtvay+
Hs-LmR
GEtvayV+
V:Hs-LmR
GEtvayV+
Hs-LmR
0
0002
0
004
0
00
6
0
00
8

08
3
35
)73(
)8
.5()04()04()4.12(
Fig. 8. Induction of luciferase gene by monopartite switches with different RXRs. (A) Plasmid DNA samples of VGEtvy + VP16:HsRXR
(HsR) or VGEtvy + VP16:Hs(1–6)-LmRXR(7–12) (V:Hs-LmR-6) or VGEtvy + VHs(1–8)-LmR(9–12) (V:Hs-LmR-8) or VGEtvy + VHs(1–9)-
LmR(10–12) (V:Hs-LmR-9) or VGEtvy + VHs(1–10)-LmR(11–12) (V:Hs-LmR-10 or VGEtvy + VHs(1–11)-LmR (12) (V:Hs-LmR-11) or VGEtvy
+ VP16:LmRXR (VLmR) and pFRLuc were transfected into 3T3 cells using superfect lipid reagent. The transfected cells were grown in
the medium containing 0, 0.008, 0.04 and 0.2 l
M concentrations of RG-102240. The cells were harvested at 48 h after adding ligand
and the reporter activity was measured and the RLU presented are mean ± SD (n ¼ 3). Numbers above the bars represent the maxi-
mum fold induction observed for that particular combination. The numbers in parentheses represent the RLU of the reporter activity in
cells that exposed to dimethylsulfoxide (no ligand). (B) Plasmid DNA samples of VGEtvy or VGEtvy + VHs-LmR or VGEtvy + Hs-LmR or
GEtvyV + VHs-LmR or GEtvyV + Hs-LmR and pFRLuc were transfected into 3T3 cells using superfect lipid reagent. The transfected
cells were grown in the medium containing 0, 0.2, 1 and 5 l
M concentrations of RG-102240. The cells were harvested at 48 h after
adding ligand and the reporter activity was measured and the RLU presented are mean ± SD (n ¼ 3). Numbers above the bars repre-
sent the maximum fold induction observed for that particular combination. The numbers in parentheses represent the RLU of the repor-
ter activity in cells that exposed to dimethylsulfoxide (no ligand). (C) The experiments and procedures used are the same as in (B)
except VGEtvay was used in place of VGEtvy. DMSO, dimethylsulfoxide.
S. K. Panguluri et al. Functional characterization of EcR gene switches
FEBS Journal 273 (2006) 5550–5563 ª 2006 The Authors Journal compilation ª 2006 FEBS 5557
GAL4:EcR fusion proteins bind to GAL4 response
element independent of ligand and RXR, but EcR
binding to HsRXR is ligand-dependent
It is not known whether GAL4:EcR (GE) fusion pro-
teins bind to GAL4 response element prior to or after
binding to ligand: ChIP assay was used to answer this

GAL4 response elements only in the presence of ligand
(Fig. 10; lane 10).
0
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0008
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DMSO
0.008
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5
DMSO
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Elam +yvtEGVyvtEGV

6.
0
0
0
7.
0
0
08.0
0
09
.
0
lortnoC E
la
m lortnoCi
A
NR Elam iANR
RXR α RXRsremirP β sremirP
Relative Expression
%7.59
%
5
.
8
7
Fig. 9. Endogenous RXR is involved in EcR gene switch function-
ing. (A) Plasmid DNA samples of VGEtvy or VGEtvy plus double-
stranded RNA for malE or VGEtvy plus double-stranded RNAs for
RXRa and RXRb and pFRLuc were transfected into HEK293 cells
using superfect lipid reagent. The transfected cells were grown in

the cells were fixed with formaldehyde at 48 h after addition of lig-
and and precipitated with VP16 antibodies as described in Experi-
mental procedures. VP16 antibodies were able to precipitate DNA–
protein complexes for all combinations tested except the GEtvy +
V:HsR treated with dimethylsulfoxide (lane 9) suggesting that
V:HsRXR binds to GEtvy only in the presence of ligand.
Functional characterization of EcR gene switches S. K. Panguluri et al.
5558 FEBS Journal 273 (2006) 5550–5563 ª 2006 The Authors Journal compilation ª 2006 FEBS
Discussion
The most significant contributions of this study are
better understanding of the molecular mechanism of
EcR gene switches in mammalian cells and develop-
ment of an efficient EcR gene switch that showed low
background activity in the absence of ligand and high
induced activity in the presence of ligand. Many of the
current EcR switches are in bipartite format, which
requires RXR for efficient transactivation of the genes
placed under their control. Among these EcR switches,
the RheoSwitch
Ò
system display the lowest back-
ground activity coupled with a strong inducibility (up
to 10 000-fold) in cultured cells [11,12]. These switches
can be very good for applications where the size of the
switch components is not a concern. However, for cer-
tain applications in which the cells are sensitive to the
slightest perturbations in the metabolic pathways
involving RXR can be a drawback [13].
In order to find the best possible monopartite switch
format, we screened different formats of switches

ligand dependent transactivation of reporter gene acti-
vation. However, the performance of the VGE format
was the best as in the case of VGE format with wild-
type EcR. GVE format showed higher background lev-
els of induction in the absence of ligand and GEV
showed low induction levels as well as low background
expression of reporter gene. The differences in the per-
formance of these monopartite switches could be due
to the difference in ligand induced conformational
changes as well as displacement of corepressors and
recruitment of coactivators by EcR fusion proteins
upon addition of ligand. The placement of EcR relat-
ive to GAL4 and VP16 probably influence the
behavior of fusion protein upon binding to ligand.
Apparently, having activation domain (VP16) and
DNA binding domain (GAL4), in that order, on the
NH
2
-terminal end of EcR is better than the other two
formats. Interestingly, this is the arrangement in nat-
ural EcRs [activation domain A ⁄ B, DNA binding
domain (C) and ligand binding (E) domain].
The difference in induction levels between cell lines
were found when we transfected VGE monopartite
switch. The difference might be due to the difference
in the levels of endogenous RXR isoforms among cell
lines. This in turn can be explained by the results
obtained in the transfection experiments with G:CfEcR
with V:RXRa or V:RXRb or V:RXRc. The data
showed that the induction levels and fold induction are

that contained a mutant CfEcR (CfEcRvy) showed
higher background activity in the absence of ligand,
resulting in only four to nine-fold inductions. To
reduce the background activity in the absence of lig-
and. Fourteen different activation domains were tested
in place of VP16 and achieved a maximum induction
of 152-fold in NIH ⁄ 3T3 and 207-fold in HEK293 cells
exposed to 1 lm RG-102240.
In this study, we also compared the performance of
two mutant EcRs with high ligand sensitivity due to
three (T335V, V390I and Y490E) or four (T335V,
V390I, A393P and Y490E) mutations in the ligand
binding domain. EcRtvy binds to both steroids and
nonsteroids while EcRtvay binds to nonsteroids but
not to ecdysteroids ([15] and data not shown). Both
these mutants performed much better than the wild-
type EcR in both bipartite and monopartite switches.
EcRtvy performed better than EcRtvay, but because
of steroid insensitivity, EcRtvay may be useful for
applications where exposure to ecdysteroids may be a
problem. EcRtvy is one of the most efficient EcR
monopartite switches developed by the present work.
The ligand induced reporter activity supported by this
switch is as good as that reported for bipartite switch
[11]. However, background activity in the absence of
ligand is slightly higher than that of bipartite switch,
which needs improvement prior to widespread use of
this format in various applications.
Ligand binding assays showed that very little
[

or GEV switches. This may be due to the fact that
these switches already use endogenous RXR and addi-
tional RXR does not improve their performance.
VP16:LmRXR and the chimeras between HsRXR and
LmRXR heterodimerize more efficiently with EcR than
does HsRXR [11], therefore these receptors are able to
improve switch performance. Our data also showed
that it is the RXR but not the additional VP16
domains present in VP16:RXR that is responsible for
improvement in the performance of these switches. The
ligand sensitivity of VGE + Hs-LmR is similar to the
sensitivity of bipartite switch, GE + V:Hs-LmRXR.
But, the induction levels of VGE + Hs-LmRXR
switch are higher when compared to those of bipartite
switch GE + V:Hs-LmRXR. The major advantages of
these new versions of VGEtvy + Hs-LmRXR and
VGEtvay + Hs-LmRXR switches are the following:
(a) because of use of mutant EcRs, ligand sensitivity of
these switches is much higher than the earlier versions
of EcR gene switches, (b) VGEtvay + Hs-LmRXR
switch can be used in applications in which potential
exposure to plant derived ecdysteroids could be a con-
cern, and (c) the removal of VP16 activation domain
from Hs-LmRXR chimera reduces the chances of inter-
ference with endogenous gene expression due to poten-
tial interaction of V:Hs-LmRXR with endogenous
nuclear receptors. The major disadvantage of this
VGEtvy + Hs-LmR switch is the slightly higher back-
ground activity in the absence of ligand when com-
pared to earlier versions of EcR bipartite switches.

designing more improved EcR gene switches in the
future.
Experimental procedures
Constructs
To make VGE, pM vector (Clonetech Inc., Palo Alto, CA,
USA) carrying DEF domains of CfEcR [9] was used as a
template to amplify GAL4:CfEcR (GE) with restriction
sites SalI on the 5¢ end and NotI on the 3¢ end, and the
PCR product was cloned into pACT vector (Promega,
Madison, WI, USA). Similarly, to make GVE, the CfEcR
was excised with restriction sites BamHI and XbaI from
pM vector and cloned in pACT vector. This pACT vector
containing EcR DEF domain was used as a template to
amplify VP16:CfEcR (VE) with restriction sites SalIon5¢
end and Not I on the 3¢ end and cloned into pBIND vector
(Promega). We have also prepared VG(93)E, VG(65)E,
G(93)VE and G(65)VE with truncated GAL4 DBD (amino
acids: 1–93 and 1–65). VGEtvy, GVEtvy, VGEtvay and
GVEtvay were prepared by replacing KpnI fragment in the
E domain of CfEcR with the KpnI fragments from CfEcR
containing T335V, V390I and Y410E mutations (tvy) and
T335V, V390I, A393P and Y410E mutations (tvay). To pre-
pare GEtvyV and GEtvayV, CfEcR DEF domains were
amplified using VGEtvy and VGEtvay as a templates using
primers containing restriction sites BamHI on the 5¢ end
and HindIII on the 3¢ end and VP16AD encoding 44 amino
scids from pVP16 with restriction sites HindIII on the
5¢ end and NotI on the 3¢ end, and these fragments
were cloned in pBIND vector (Promega). Construction of
VP16:HsRXR (a fusion between VP16 activation domain

A second reporter, Renilla luciferase, expressed under a
thymidine kinase constitutive promoter was cotransfec-
ted into cells and used for normalization. After 4 h of
transfection, 125 lL of DMEM containing 20% fetal
bovine serum and double the concentration of inducer,
RG-102240 also known as GS
TM
-E or RSL 1 was added.
Two days after transfection, the medium was discarded
and the cells were lysed in 50 lL passive lysis buffer
(Promega). Twenty microliters of extract were transferred
to 96-well opaque plates and the luciferase and Renilla
luciferase reporter activities were measured using the Dual
luciferase
TM
reporter assay system from Promega and
Fluoroskan Ascent FL (ThermoLab Systems, Helsinki,
Finland). All the transfection experiments were performed
in triplicate and the experiments were repeated at least
three times.
Ligand binding
To determine whether CfEcR in VGE, GVE and GEV
(with EcRtvy and EcRtvay mutants) format, needs heterod-
imeric partner to bind to ligand, VGE, GVE and GEV con-
structs were cloned in pGEX vector (Promega) in the sites
SalI and NotI and expressed in BL-21 Escherichia coli cells.
Fifteen milliliters of LB medium containing 50 lg ÆL
)1
amp-
icillin was inoculated and grown overnight at 30 °C. The

)1
and 99% pure (HPLC purified) is competed
with 10 mm RG-102240. A similar reaction mixture was
made for Scatchard analysis with 1 lL of different dilu-
tions of [
3
H]RH-112485. The reaction mixture was kept
at 4 °C for 4 h; the unbound [
3
H]RH-2485 was removed
by activated charcoal:dextran (10 : 1) and the bound
radioactivity was measured in liquid scintillation counter.
The background radioactivity was measured by adding all
the ingredients except proteins and was deducted from
the hot and cold values. The counts for liquid scintilla-
tion cocktail were also measured and were taken as refer-
ence or blank. The counts of the radioactive ligand alone
in the scintillation liquid cocktail were also measured and
these values were taken in the y-axis as total added. The
reactions were done in triplicate. The dissociation con-
stant, K
d
, was evaluated from the saturation curve for
radioligand binding using nonlinear regression analysis
with one site binding (hyperbola) equation in the graph-
pad prism program (GraphPad Software, San Diego, CA,
USA).
ChIP assay
To determine whether the receptor proteins need ligand to
bind to response element, ChIP assay was performed by

RNAi
RNAi experiments were performed in HEK293 cells trans-
fected with double-strand RNA for RXRa and RXRb to
determine whether endogenous RXR is helping the mono-
partite gene switches in the transactivation of reporter
gene. HEK293 cells were transfected at 40% confluence
with 8 lg each of RXRa and RXRb double-stranded
RNAs per well in 12-well plate, and the cells were harves-
ted three days after transfection. Bacterial malE gene dou-
ble-stranded RNA was used as a control. Total RNA was
isolated using Trizol reagent (Molecular Research Center
Inc., Cincinnati, OH, USA). First strand cDNA was pre-
pared by using M-MuLV Reverse Transcriptase (New Eng-
land Biolabs, Ipswich, MA, USA). Quantitative PCR
analysis was performed to determine whether the endog-
enous RXR was knockdown or not. Monopartite gene
switch VGEtvy, pFRLuc and double-stranded RNA for
RXRa and RXRb were transfected. The transfected cells
were exposed to ligand for 48 h and the luciferase activity
was measured.
Acknowledgements
This work was supported in part by RheoGene Inc.
research grant to SRP. This is contribution number
06-08-115 from the Kentucky Agricultural Experimen-
tal Station. We thank Dr Dean Cress of RheoGene
Inc. for support throughout the study and critical
reading of the manuscript.
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