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Inducibles_1&2

DEACTIVATOR SYSTEMS: POFF and EOFF
T r 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) trans-genes gives the possibility to modulate the expression of a given Gene Of Interest (GOI).
The EFFECTOR-mouse expresses an Oper-ator-Binding Protein (OBP) which can be linked to further functional proteins,for example to a trans-activation (e.g.
harbour the GOI and a (e.g. CMV-derived)minimal promoter (Pmin), combined withOperator (O) DNA sequences, to whichthe OBP has binding affinity.
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
As demonstrated in CHO cell culture experiments, GOI expression is reduced by approximately100-fold over respective antibiotic concentrations ranging from roughly 0.01 to 2 µg/ml1,2. 1000 10000
Antibiotic (ng/ml)
Reversibility
The responsiveness of SEAP producing EOFF cells to recurrent addition and withdrawal of Erythro-mycin reveals a swift and efficient on/off state transition2.
Split cell cultures adjusted to 105 cells/ml points (circled). Erythromycin (2 µg/ml) and added again at t = 90 hrs (black).
Culture with inversed Erythromycin status: Independent Control
As demonstrated by interference studies on CHO cells co-transfected with pairs of EFFECTOR andRESPONDER elements, gene expression modulation is achieved with the system-compatible antibiotic, only2.
Pristina-
Erythro-
Pristina-
Erythro-
Antibiotic
Antibiotic
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 perspectivesin terms of multi-gene and multi-regulated interventions3, 4. Positioning
To be provided with custom-made RESPONDER mice, PolyGene will maintain various EFFECTOR mouselines constitutively expressing the fused OBP at determined levels. Thus, mating of RESPONDER micewith different EFFECTORS (each of a given trans-activation potential) allows studying GOI expressionbeyond 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, info@polygene.ch, www.polygene.ch

Source: http://www.polygene.ch/pdf/Inducibles2.pdf

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