Tet-On® 3G Inducible Expression System
Mammalian Expression Systems
Lowest background, highest sensitivity
The Tet-On 3G Tetracycline Inducible Gene Expression Systems are the 3rd generation of the most
powerful, versatile, and widely cited inducible mammalian expression systems available. They provide precisely
regulated control of transgene expression that is reversible, quantitative, and reproducible. The 3G system
offers a significant improvement over the exquisite Tet System technologies developed in the laboratories
of Hermann Bujard, Manfred Gossen, and Wolfgang Hillen (1, 2) by combining a new promoter that shows
significantly reduced background (3), and a new transactivator protein with increased sensitivity (4).
How Do Tet-On 3G Inducible Systems Work?
Target cells that express the Tet-On 3G transactivator protein and contain a gene of interest (GOI) under the control of a TRE3G
promoter (PTRE3G) will express high levels of your GOI, but only when cultured in the presence of doxycycline (Dox), a tetracycline
analog. When bound by Dox, the Tet-On 3G protein undergoes a conformational change that allows it to bind to tet operator (tetO)
sequences located in PTRE3G (Figure 1). Unlike inferior TetR-based systems (never sold by Clontech), Tet-On technologies activate
rather than repress transcription, a critical difference which results in far lower basal expression, higher maximal expression, a more
rapid response time—and ultimately, a system proven to be the first choice for conditional expression in vivo.
No Dox
Tet-On 3G
Transactivator
PTRE3G
Promoter
Dox
Doxycycline
Transcription
Figure 1. The Tet-On 3G systems allow inducible gene expression only in the presence of doxycycline. When Dox binds, the transactivator undergoes
a conformational change allowing it to bind tet operator (tetO) repeats within the TREG Promoter (PTRE3G). The transactivator activates expression
through transcription activation domain repeats.
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Tet-On® 3G
…continued
Lowest-Ever Background, Highest Sensitivity
The combination of the following two optimized elements makes Tet-On 3G better than previous generations of the Tet-On system
(Figures 2 & 5, Table I):
1. PTRE3G promoter—mutations have reduced background expression from the inducible promoter by up to 20-fold (Figure 2).
2. Tet-On 3G transactivator protein—mutations have significantly increased its sensitivity to the inducer doxycycline (Dox),
so that at very low Dox concentrations (between 5–10 ng/ml), expression can be 100–150 fold higher when using a Tet-On 3G system
compared to Tet-On Advanced (Figure 3).
When the two elements are combined, not only can you detect very high expression of your protein after exposure to just 10 ng/ml
of Dox (Figure 4), but the background is so low that even in transient cotransfections we have seen up to a 27,000-fold difference
in expression between the induced and uninduced state (MCF7 cells, data not shown).
160,000
80,000
Tet-On
Advanced
Tet-On 3G
Reduced
Background
600
0
Tet-On 3G
12,000
8,000
4,000
40,000
0
Transactivator
Tet-On
Advanced
RLU
RLU
120,000
16,000
System
800
0
50
ng/ml
0
500
0
5
10
50
Doxycycline concentration (ng/ml)
100
Figure 2. The PTRE3G promoter results in significantly reduced basal expression.
HEK 293 cells were transiently cotransfected with both the response vectors
(containing luciferase) and regulator vectors from each of the Tet-On 3G
and Tet-On Advanced Inducible Expression Systems. The cells were cultured
in the presence and absence of Dox, and after 24 hr, luciferase expression
was measured. Although both systems provided strong expression in the
presence of Dox, the Tet-On 3G System produces far lower background
expression in the absence of Dox (inset).
Figure 3. Tet-On 3G demonstrates higher sensitivity to doxycycline than
Tet-On Advanced. Tet-On 3G and Tet-On Advanced genes were integrated
at the same locus in a stable HLF33 cell line expressing luciferase from a
TRE promoter. For each of these two double-stable cell lines, induced luciferase
expression was measured in response to a range of doxycycline (Dox)
concentrations. At 5–10 ng/ml Dox, induced expression was 100–150-fold
higher for the Tet-On 3G cell line, and at 50 ng/ml, expression was 4.6 fold
higher (data kindly provided by Professor W. Hillen and Dr. C. Berens,
University of Erlangen).
A
B
kD
0
10–1
Dox (ng/ml)
10 0
101
102
103
50–
pTRE3G-Luc
160,000
120,000
Luciferase
RLU
70–
b-Actin
80,000
40,000
0
0
10–¹
100
10¹
10²
10³
ng/ml
Figure 4. Tet-On 3G is highly sensitive to as little as 10 ng/ml of doxycycline. Following cotransient transfection of pCMV-Tet3G and pTRE3G-Luc in HeLa
cells, increasing levels of Dox were added and expression of luciferase was measured using an anti-luciferase antibody (Panel A) and a luciferase assay (Panel B).
Induced expression was very high even with Dox concentrations as low as 10 ng/ml, and was detectable even at 0.1 ng/ml Dox.
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Tet-On® 3G
…continued
The Tet-On 3G System is a Powerful Combination
of Two Elements Optimized by Mutation
Tet-On 3G Transactivator Protein
Tet-On 3G is a modified form of an improved Tet-On Advanced transactivator protein which has been evolved to display far higher
sensitivity to doxycycline (4). The protein consists of a modified bacterial Tet repressor (TetR) fused to three minimal VP16 activation
domains to create a transcriptional activator protein (transactivator). Five amino acid changes convert Clontech’s Tet-On Advanced
transactivator to Tet-On 3G (for a comparison of the various Tet-On transactivator sequences, see www.clontech.com). Tet-On 3G
generates very high maximal expression and responds to lower Dox concentrations than its predecessors. These Dox concentrations
are far below cytotoxic levels for either cell culture or transgenic studies. The increased Dox sensitivity is particularly advantageous
for in vivo studies in tissues where high Dox concentrations are difficult to attain (e.g., brain).
Table I: Three Generations of Tet-On Inducible Expression Systems
Generation
Transactivator Protein
Inducible Promoter
Tet-On System
Name
1st
Tet-On
PTRE2
Tet-On Advanced System
2nd
Tet-On Advanced
PTIGHT
Tet-On 3G System
3rd
Tet-On 3G
PTRE3G
HEK 293 Cells
HeLa Cells
1,600
8,000
Tet-On
Tet-On Advanced
Fold induction
Tet-On 3G
1,200
6,000
800
4,000
400
2,000
0
1st
2nd
Generation
3rd
0
1st
2nd
Generation
3rd
Figure 5. Comparison of the three generations of Tet-On. Panel A shows the transactivator and inducible promoter combinations for each generation
of the Tet-On system. Tet-On was launched by Clontech in 1996—at the time this was the premier inducible expression system, and its performance has
only been surpassed by subsequent generations of the Tet-On system. Panel B shows co-transient transfection experiments with both vectors in HEK 293
and HeLa cells. Although Tet-On Advanced shows great improvement over the original Tet-On System when comparing the difference between the induced
and the uninduced states (fold induction), Tet-On 3G shows even higher fold induction due to the significantly reduced basal expression provided by
the PTRE3G promoter.
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Tet-On® 3G
…continued
PTRE3G Inducible Promoter
The inducible promoter PTRE3G provides for very low basal expression and high maximal expression after induction (3). It consists of 7
repeats of a 19 bp tet operator sequence located upstream of a minimal CMV promoter, which we refer to as the tetracycline response
element (TRE). Although the sequences of tet operator repeats are identical in all Tet-On generations (Figure 6), the junction sequences
of PTRE3G have been altered to an even spacing and the central portions are randomized. Additionally, elements from the minimal
CMV promoter have been mutated to consensus.
Because PTRE3G lacks binding sites for endogenous mammalian transcription factors and is inactive in the absence of a transactivator
protein, basal expression from PTRE3G is the lowest of any TRE-containing promoter available so far.
TRE Promoter Alignment
tetO
tetO
T TTACCACTCCCTATCAGTGATAGAGAAAAGTGAAAGTCGAGTTTACCACTCCCTATCAGTGATAGAGAAAA-GTGAAAGTC
PTRE2
(1)
PTight
(1)
PTRE3G
(1)
T TT---ACTCCCTATCAGTGATAGAGAAC---GTATGAAGAGTTTA---CTCCCTATCAGTGATAGAGAACGTATG----CA
PTRE2
(82)
PTight
G AGTTTACCACTCCCTATCAGTGATAGAGAAAAGTGAAAGTCGAGTTTACCACTCCCTATCAGTGATAGAGAAAAGTGAAAG
(69)
T TT---ACTCCCTATCAGTGATAGAGAAC---GTATGTCGAGTTTA---CTCCCTATCAGTGATAGAGAACG-ATG----TC
tetO
tetO
G AGTTTAC---TCCCTATCAGTGATAGAGAAC---GTATGTCGAGTTTA---CTCCCTATCAGTGATAGAGAAC---GTATG
PTRE3G (70) G A C T T T A C - - - T C C C T A T C A G T G A T A G A G A A C - - - G T A T A A G G A G T T T A - - - C T C C C T A T C A G T G A T A G A G A A C - - - G T A T G
tetO
tetO
PTRE2 (164) T C G A G T T T A C C A C T C C C T A T C A G T G A T A G A G A A A A G T G A A A G T C G A G T T T A C C A C T C C C T A T C A G T G A T A G A G A A A A G T G A A
PTight (139) T C G A G T T T A C - - - T C C C T A T C A G T G A T A G A G A A C - - - G T A T G T C G A G T T T A - - - - T C C C T A T C A G T G A T A G A G A A C - - - G T A
PTRE3G (140) A C C A G T T T A C - - - T C C C T A T C A G T G A T A G A G A A C - - - G T A T C T A C A G T T T A - - - C T C C C T A T C A G T G A T A G A G A A C - - - G T A
tetO
Modified Minimal CMV Promoter
PTRE2 (246) A G T C G A G T T T A C C A C T C C C T A T C A G T G A T A G A G A A A A G T G A A A G T C G A G C T C G G T A C C C G G G T C G A G G T A G G C G T G T A C G G T
PTight (208) T G T C G A G T T T A C - - - T C C C T A T C A G T G A T A G A G A A - - C - - - - - - - - - - - - - - - G T A T - - - - G T C G A G G T A G G C G T G T A C G G T
PTRE3G (210) T A T C C A G T T T A C - - - T C C C T A T C A G T G A T A G A G A A - - - - - - - - - - - - C - - - - - G T A T - - - - - A A G C T T T A G G C G T G T A C G G T
Modified Minimal CMV Promoter (cont.)
PTRE2 (328) G G G A G G C C T A T A T A A G C A G A G C T C G T T T A G T G A A C C G T C A G A T C G C C T G G A G A C G C C A T C C A C G C T G T T T T G A C C T C C A T A G
PTight (266) G G G A G G C C T A T A T A A G C A G A G C T C G T T T A G T G A A C C G T C A G A T C G C C T G G A - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -
PTRE3G (267) G G G C G - C C T A T A A A A G C A G A G C T C G T T T A G T G A A C C G T C A G A T C G C C T G G A G C A A T - - T C C A C A A C A C T T T T G T C T T A T A C C
PTRE2 (410) A A G A C A C C G G G A C C G A T C C - - - - - - - - - - - PTight (317) - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - PTRE3G (346) A A C T T T C C G T A C C A C T T C C T A C C C T C G T A A A
Figure 6. Sequence comparison of the three generations of TRE promoters sold by Clontech. Each promoter consists of 7 identical repeats of the tet operator
sequence (green), and contains a minimal CMV promoter—although this sequence has been mutated to consensus in PTRE3G (3). Moreover, in PTRE3G the spacer
sequences between the tetO repeats are evenly spaced and contain randomized central sequences.
Combining these two components (the PTRE3G promoter and Tet-On 3G transactivator) results in the largest difference between the induced
and uninduced states (fold induction) of any commercially available inducible expression system for mammalian cells (Figure 5).
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Tet-On® 3G
…continued
Choice of Tet-On 3G Vector Formats
Figure 7 shows the range of Tet-On 3G vector formats available. Each is sold as a complete system that includes:
• a Tet-On transactivator vector (either pCMV-Tet3G or pEF1a-Tet3G)
• a TRE response vector with a PTRE3G promoter and a multiple cloning site
• both hygromycin and puromycin linear selection markers
• Tet Approved FBS
• Xfect™ transfection reagent
Transactivator Vectors
pCMV-Tet3G expresses the Tet-On 3G protein constitutively from a CMV promoter, and is included in all Tet-On 3G systems
except the EF1 alpha version, in which an EF-1 alpha promoter expresses the transactivator.
pCMV-Tet3G
PCMV IE
Tet-On 3G
PSV40
Neor
Tet-On 3G
PSV40
Neor
pEF1a-Tet3G
PEF-1a
Response Vectors
All response vectors contain a PTRE3G promoter. pTRE3G is included in both the core system and the EF-1 alpha version.
pTRE3G-IRES can inducibly coexpress any two genes of interest, and is included with the bicistronic Tet-On 3G system.
Alternatively, you can monitor inducibility using red or green fluorescent proteins if you are using the mCherry or ZsGreen1 systems.
PTRE3G
MCS
pTRE3G
pA
MCS
PTRE3G
MCS
pTRE3G-IRES
IRES
pA
IRES
MCS
PTRE3G mCherry
pA
IRES
MCS
pTRE3G-mCherry
pA
pTRE3G-ZsGreen1
PTRE3G ZsGreen1
Linear Selection Markers
Selection for inducible clones can be performed using either hygromycin or puromycin linear selection markers.
Both markers are included with every Tet-On 3G system.
Linear Hygromycin Marker
PSV40
Hygr
pA
Linear Puromycin Marker
PSV40
Puror
pA
Figure 7. Vector formats for Tet-On 3G systems. For complete components lists, see www.clontech.com
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Tet-On® 3G
…continued
Inducible Expression in Stem Cells and Hematopoietic Cells
The Tet-On 3G Inducible Expression System (EF1a Version), our EF-1 alpha promoter version of the Tet-On 3G system, provides
for consistent long-term expression of the Tet-On 3G transactivator, even in cell types known for their tendency to silence a CMV
promoter over time, such as hematopoietic cells and stem cells (Figure 7). We tested the EF-1 alpha version in Jurkat cells, a cell line
known to show reduced expression and clonal variation in expression from CMV- based vectors. When expressing the Tet-On 3G
transactivator protein from the EF-1 alpha promoter, 83% of the Jurkat Tet-On 3G clones showed strong inducible expression
and 33% demonstrated very high inducibility (greater than 2,000-fold). Such levels of control are not possible when using previous
versions of the Tet-On system for this cell line (Figure 8).
The EF-1 alpha promoter is a preferred promoter for gene expression in stem cells. We demonstrated that the Tet-On 3G
Inducible Expression System (EF1a version) displays excellent performance in mouse embryonic stem cells (Figure 9).
Jurkat Tet-On 3G Cells
200,000
– Dox
+ Dox
160,000
RLU
120,000
80,000
40,000
0
1
2
3
4
5
6
7
8
9
10
Clone no.
11
12
13
14
15
16
17
18
Figure 8. Tet-On 3G (EF1α Version) provides inducible expression in hematopoietic cells. Jurkat cells were transfected with pEF1α-Tet3G using Xfect
transfection reagent, and stable clones were selected by limiting dilution. 18 stable clones were then tested for inducibility by transient transfection
using pTRE3G-expressing luciferase. Six out of eighteen clones showed more than 2,000-fold induction via transient transfection.
Mouse Embryonic Stem Cells
Dox
No Dox
A
B
C
D
Figure 9. Tet-On 3G (EF1α Version) provides inducible expression in stem cells. Mouse embryonic stem cells (ES-E14TG2a mES cells) were cotransfected with
pTRE3G-ZsGreen1 and pEF1α-Tet3G using Xfect Stem transfection reagent. The stem cells show ZsGreen1 expression only in the presence of Dox (Panel C).
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Tet-On® 3G
…continued
Induce Expression of Two Genes Simultaneously
with IR ES Bicistronic Vectors
The Tet-On 3G Inducible Expression System (Bicistronic Version) allows the simultaneous expression of two genes of interest
using bicistronic vectors. The key element of these vectors is an optimized internal ribosome entry site (IRES) flanked by two MCSs.
The inclusion of the IRES permits two genes of interest to be coexpresssed as separate proteins from a single mRNA transcript.
Although translation initiation of eukaryotic mRNAs occurs almost exclusively at the 5' cap, the IRES allows ribosomes to bind and
initiate translation at a second, internal location. Thus, two proteins are expressed simultaneously from a single bicistronic mRNA
transcript (Figures 10 and 11).
Select for Clones and Monitor Inducible Expression
using Bright Fluorescent Proteins
PTRE3G
IRES
MCS
A
MCS
Using the same IRES technology, we’ve married very tight gene expression control to a bright red fluorescent protein (mCherry)
in the Tet-On 3G Inducible Expression System (with mCherry) and a bright green fluorescent protein (ZsGreen1) in the Tet-On 3G
Inducible Expression System (with ZsGreen1). If your cells turn red or green after adding Dox, this confirms that your gene
has been turned on and that you have selected a high-performing inducible clone (Figure 11).
C
pA
Luciferase expression from MCS II
500,000
mCherry
Luciferase
400,000
B
Expression of mCherry from MCS I
0 ng/ml
10–1 ng/ml
100 ng/ml
RLU
300,000
200,000
101 ng/ml
102 ng/ml
103 ng/ml
100,000
0
0
10–¹
100
10¹
10²
10³
ng/ml
Figure 10. Co-inducible expression of two genes from a single bicistronic transcript. pTRE3G-IRES expressing both mCherry, from MCS I, and luciferase,
from MCS II (Panel A), was cotransfected with pCMV-Tet3G into HeLa cells with increasing levels of Dox. Induced expression of mCherry was monitored
by fluorescent microscopy (Panel B) and luciferase expression was measured using a luciferase assay (Panel C).
mRNA
Doxycycline
Ribosome
Transcription
PTRE 3G
mCherry
IRES
Your gene
pA
5' end
mCherry
Fluorescent protein
IRES
Your gene
AAAAAA
Protein of interest
Figure 11. Monitor inducible expression and easily screen for inducible clones using bright fluorescent proteins. Clones created using the Tet-On 3G
Inducible Expression System (with mCherry) or Tet-On 3G Inducible Expression System (with ZsGreen1) will respectively fluoresce red and green, but only
in the presence of Dox. The size (bp) of the fluorescent protein gene is optimal for maximum expression of your GOI from the internal ribosome entry site (IRES).
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7
Tet-On® 3G
Mammalian Expression Systems
…continued
Use Tetracycline Approved Serum for Optimal Results
With the greatly increased sensitivity of the Tet-On 3G system, it is more important than ever that the fetal bovine serum you use
for your studies is guaranteed to be tetracycline-free (Figure 12). Only Clontech performs actual inducibility tests on a sensitive
Tet inducible cell line in order to provide an absolute guarantee that your serum will not affect basal expression in your Tet-On 3G
experiments). Each Tet-On 3G system is supplied with 50 ml of our premium Tet-Approved FBS.
Visit
Visit
sit our website
for more details!
click here…
Figure 12. To ensure low background with Tet-On 3G, it is essential to use fetal bovine serum that is functionally tested. Clontech offers four such serum
options, including serum that is sourced from the US, Mexico, and Australia, as well as US-sourced serum that is additionally tested for use in mouse
embryonic stem cells.
References
1.
2.
3.
4.
Gossen, M. & Bujard, H. (1992) Proc. Natl. Acad. Sci. USA 89(12):5547–5551.
Urlinger, S. et al. (2000) Proc. Natl. Acad. Sci. USA 97(14):7963–7968.
Löw, R., Heinz, N., Hampf, M., Bujard, H. & Gossen, M. (2010) BMC Biotechnology 10:81.
Zhou, X., Vink, M., Klaver, B., Berkhout, B. & Das, A. T. Optimization of the Tet-On system for regulated gene expression through viral evolution. (2006)
Gene Ther. 13(19):1382–1390.
Ordering Information
Product
Size
Cat. No.
Tet-On 3G Inducible Expression System
each
631168
Tet-On 3G Inducible Expression System (EF1α Version)
each
631167
Tet-On 3G Inducible Expression System (Bicistronic Version)
each
631166
Tet-On 3G Inducible Expression System (with mCherry)
each
631165
Tet-On 3G Inducible Expression System (with ZsGreen1)
each
631164
Tet System Approved FBS
50 ml
500 ml
631107
631106
Doxycycline
5g
631311
TetR Monoclonal Antibody (Clone 9G9)
40 µg
200 µg
631131
631132
Notice to Purchaser
For all licensing information, visit www.clontech.com
Clontech Laboratories, Inc.
A Takara Bio Company
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