INDUCIBLES 1
DEREPRESSOR SYSTEMS: PON and EON
Tr a n s g e n e A c t i v a t i o n w i t h A n t i b i o t i c s
Two novel systems for independent modulation of transgene expression in mice, i.e. Pristinamycininducible (PON or PipON)1 and Erythromycin-inducible (EON)2 set-ups, are now available at PolyGene.
Mode of Function
These unique systems, developed at the Swiss Federal Institute of Technology in Zurich, are based on the
capacity of antibiotics to bind repressor molecules and thus their capacity to modulate gene expression.
Crossing of two complementary (EFFECTOR and RESPONDER) transgenic mouse lines yields offspring,
in which expression of the Gene Of Interest (GOI) is actively inhibited.
No Antibiotic:
With Antibiotic:
The EFFECTOR-mouse expresses an Operator-Binding Protein (OBP) which can be
linked to further functional proteins,
for example to a trans-silencing (e.g.
KRAB) domain as represented here (Stop).
OBP
Stop
OBP
Stop
P
O
GOI
P
O
GOI
The RESPONDER-mouse is engineered to
harbour the GOI and a functional Promoter (P), combined with Operator (O)
DNA sequences, to which the OBP has
binding affinity.
Transcription is de-repressed by administration of the respective antibiotic, which dissociates the
fused-Operator Binding Protein (OBP) from the Operator. GOI expression is thus unleashed and
free to be driven by a definable (e.g. tissue specific) Promoter.
Dose-Response
While binding of OBP alone strongly reduces GOI expression, it is literally abolished when
KRAB-OBP fusion protein is bound to the operator sequences1.
Expression, as shown in cell culture experiments, is gradually increased up to roughly 100-fold at
antibiotic concentrations ranging from about 0.1 to 2 µg/ml1,2.
As evidenced in vitro, protein production follows linear kinetics over a period of approx. 70 hours2.
Bioavailability
Streptogramins (e.g. Pristinamycin) and Macrolides (e.g. Erythromycin), many of which have had a
proven track record as antibiotic agents for over half a century, are reputed for excellent bioavailability and optimal pharmacokinetics. The low concentrations required for adjusting responsive
elements are reached in all tissues following as well intravenous as oral administration.
INDUCIBLES 1
System Compatibility
Serum EPO (U/I)
Tested on mice with implanted HT-1080 cell derivatives, the EON system was also proven to respond
in vivo, in a dose-dependent manner2.
60
50
40
30
20
10
10
Microencapsulated cells producing Erythropoietin (EPO) under
Erythromycin control were implanted in the peritoneal cavity of
receptor mice. EPO synthesis was monitored 72 hrs after injection
of Erythromycin at indicated doses.
Ctrl
0
0.1
1
10
Non-transplanted control mice (Ctrl), irrespective of the quantities
of antibiotic administered, showed endogenous EPO production
levels at the detection limit.
Erythromycin (mg/kg)
Independent Control
As evidenced in antibiotic interference studies2, the different systems are fully compatible, thus
paving the way for independent regulation of different transgenes in a single cell or combinatorial
transcription control3.
Positioning
PolyGene offers free promoter choice in RESPONDER constructs, thus ideally positioning both
(PON and EON) DEREPRESSOR systems for tissue specific applications.
Note that the initial RESPONDER mouse, exhibiting promoter-driven GOI expression, can serve as
constitutive EXPRESSOR. Caution is however warranted for transgenes affecting embryogenesis.
Hence, the DEREPRESSOR systems are best suited for transgene expression modulation in adult mice.
Streptogramins as well as Macrolides are known for their low interference with host metabolism.
Apart from possible concerns in the field of immunology (with PON as preferred option), or in that
of neurology (for which EON may seem safer), both the PON and the EON systems are indicated for
research applications in a broad range of therapeutic areas.
References:
1. Fussenegger, M. et al., Streptogramin-based gene regulation systems for mammalian cells. Nat. Biotechnol. 18, 1203-1208 (2000)
2. Weber, W. et al., Macrolide-based transgene control in mammalian cells and mice. Nat. Biotechnol. 20, 901-906 (2002)
3. Kramer, B. et al., BioLogic Gates Enable Logical Transcription Control in Mammalian Cells. Biotechnol. Bioeng. 87, 478-484 (2002)
PolyGene AG, Riedmattstrasse 9, CH - 8153 Rümlang, Phone +41 44 828 63 80, Fax +41 44 828 63 81, [email protected], www.polygene.ch
INDUCIBLES 2
DEACTIVATOR SYSTEMS: POFF and EOFF
Tr a n s g e n e S u p p r e s s i o n w i t h A n t i b i o t i c s
PolyGene's Pristinamycin- and Erythromycin-inducible expression systems are also available as
DEACTIVATORS, i.e. POFF and EOFF, respectively1,2.
Mode of Function
In their POFF and EOFF versions, the co-expression of two distinct (EFFECTOR and RESPONDER) transgenes gives the possibility to modulate the expression of a given Gene Of Interest (GOI).
No Antibiotic:
With Antibiotic:
The EFFECTOR-mouse expresses an Operator-Binding Protein (OBP) which can be
linked to further functional proteins,
for example to a trans-activation (e.g.
VP16) domain as represented here (Go).
OBP
Go
OBP
The RESPONDER-mouse is engineered to
harbour the GOI and a (e.g. CMV-derived)
minimal promoter (Pmin), combined with
Operator (O) DNA sequences, to which
the OBP has binding affinity.
Go
O
Pmin
GOI
O
Pmin
GOI
Transcription is de-activated by administration of the respective antibiotic, dissociating OBP from
Operator and thus depriving the GOI of a functional activator.
Dose-Response
SEAP Production
(U/l)
As demonstrated in CHO cell culture experiments, GOI expression is reduced by approximately
100-fold over respective antibiotic concentrations ranging from roughly 0.01 to 2 µg/ml1,2.
80
POFF
EOFF
60
40
20
10
0
1
10
100
1000 10000
Antibiotic (ng/ml)
Reversibility
SEAP (U/l)
The responsiveness of SEAP producing EOFF cells to recurrent addition and withdrawal of Erythromycin reveals a swift and efficient on/off state transition2.
18
15
12
9
6
3
0
hrs
0
17
27
39
40
66
73
89
90
113
119
143
Split cell cultures adjusted to 105 cells/ml
in fresh medium at the indicated timepoints (circled). Erythromycin (2 µg/ml)
was added at t = 0, not added at t = 40
and added again at t = 90 hrs (black).
Culture with inversed Erythromycin status:
pink.
INDUCIBLES 2
Independent Control
As demonstrated by interference studies on CHO cells co-transfected with pairs of EFFECTOR and
RESPONDER elements, gene expression modulation is achieved with the system-compatible antibiotic, only2.
POFF
Pristinamycin
Erythromycin
Tetracycline
EOFF
No
Antibiotic
Pristinamycin
Erythromycin
Tetracycline
No
Antibiotic
30
SEAP Production
(U/l)
25
20
15
10
5
0
System Compatibility in vivo
In mice, the EOFF system was proven to respond in a dose-dependent fashion2. Moreover, the
availability of different systems for independent control of transgene expression opens perspectives
in terms of multi-gene and multi-regulated interventions3, 4.
Positioning
To be provided with custom-made RESPONDER mice, PolyGene will maintain various EFFECTOR mouse
lines constitutively expressing the fused OBP at determined levels. Thus, mating of RESPONDER mice
with different EFFECTORS (each of a given trans-activation potential) allows studying GOI expression
beyond a 2-log range.
Since the initial (OFF-type) RESPONDER mouse does not express the GOI, DEACTIVATOR systems are
also suited for investigating embryonic lethal genes.
The reputed excellence of both, Streptogramins and Macrolides, in terms of bioavailability and of
low interference with host metabolism, distinguishes the POFF and EOFF systems for a variety of
applications, including for pregnancy-related studies.
References:
1. Fussenegger, M. et al., Streptogramin-based gene regulation systems for mammalian cells. Nat. Biotechnol. 18, 1203-1208 (2000)
2. Weber, W. et al., Macrolide-based transgene control in mammalian cells and mice. Nat. Biotechnol. 20, 901-906 (2002)
3. Fux, C. et al., Streptogramin- and tetracycline-responsive dual regulated expression of p27Kip1 sense and antisense enables
positive and negative growth control of Chinese hamster ovary cells. Nucleic Acids Res. 29, e19 (2001)
4. Kramer, B. et al., An engineered epigenetic switch in mammalian cells. Nat. Biotechnol. 22, 867-870 (2004)
PolyGene AG, Riedmattstrasse 9, CH - 8153 Rümlang, Phone +41 44 828 63 80, Fax +41 44 828 63 81, [email protected], www.polygene.ch