Regulation via Hormones - Customized Transgenic Mouse and Rat Models

Protein activity modulation with RU486.

Starting from the mutant rat glucocorticoid receptor by Prof. Sandro Rusconi (University of Fribourg, Switzerland), PolyGene has elaborated an enhanced RuX system with versatile and powerful properties for sensitive gene regulation in tissue culture and in vivo.

Mode of Function

The RuX system is based on a mutant Glucocorticoid-Receptor Ligand-Binding-Domain (GRLBD), which is silent with natural or synthetic glucocorticoids but strongly activated by RU486 (mifepristone, Lanz et al, 1994).

As opposed to sex hormone receptors, the glucocorticoid receptor (GR) is mainly localized to the cytoplasm in hormone-free cells. Ligand binding triggers the translocation of GR through the nuclear
pore complex and, when bound to specific target sites within the chromatin, hormone-GR complexes influence the transcriptional activity of linked promoters (Pratt et al, 1993, DeFranco, 2002).

ON-type inducible transgene expression system: Fused to the RuX GRLBD in the effector element, the represented heterologous moieties comprise a trans-activating domain (VP16) to activate expression of a Gene Of Interest (GOI), and a yeast-derived Gal4 domain with high affinity for the Gal4-DNA binding site (GBS). With RU486, the constitutively expressed (and otherwise cytoplasm borne) chimeric effector protein is activated and translocated to the cell nucleus. In this configuration, binding of the effector molecule to the GBS of the responder element induces minimal promoter (P^min)-driven expression of the GOI.

Breeding of complementary transgenic (EFFECTOR & RESPONDER) RuX mouse lines yields offspring, in which expression of a given Gene Of Interest (GOI) is induced with RU486.

Key features of this approach to regulating protein activity are unique in combination. On fusion of heterologous domains to GRLBD, it can function as a regulatory cassette and subject various protein functions to hormonal control (Picard et al, 1991).

RU486 triggers migration of GRLBD-fusion protein from the cytoplasm into the nucleus. A DNA construct harbouring GRLBD and green fluorescing protein (GFP) gene sequences was transfected into HER911 cells.
Left: Hormone-free culture, Right: With RU486

Dose Response

The RuX system allows gradual activation of GOI expression to over 1000-fold, at sub-pharmacological doses of RU486.

A trans-activating effector construct was co-transfected into HeLa cells with a Gaussia luciferase reporter plasmid. Cells were cultured in hormone-free medium supplemented with RU486 or dexamethasone (Dex) at the indicated concentrations. Luciferase activity was monitored with a luminometer after 24 hours.

Dexamethasone, the potent synthetic ligand for the wild-type glucocorticoid receptor, does not affect expression levels (as evidenced by Gaussia luciferase activity at the detection limit through highest concentrations).

Compatibility in mice

As demonstrated with a RuX-like system, RU486 (e.g. orally administered) induces transgene expression in a dose-dependent manner. Expression levels peak at 10nM RU486 which is about 1000 times less than the lowest toxical dosis (TDLO) in mouse, within approximately 10-12 hours (Wang et al, 1996). Under those conditions, precise temporal control is achieved, as a consequence of the rapid RU486 induction kinetics. Transgene expression returns to undetectable levels upon ligand withdrawal, and is re-inducible upon administration of the ligand.

Positioning

Based on the excellent properties with increased activation ratio and reduced background levels, the enhanced RuX system can be used for a variety of applications in non-toxic, reversible transgenic gene regulation.
The strict nuclear translocation upon hormone binding of the glucocorticoid receptor also offers a unique way to configure cytoplasm-nucleus shuttling systems (e.g. the shuttling and activation of site-specific recombinases, or of marker peptides).

References

Lanz, B. and Rusconi, S.,
A conserved carboxy-terminal subdomain is Important for ligand interpretation and trans-activation by nuclear receptors.
Endocrinology 135 Nr 5, 2183-2195 (1994).

Pratt, W. B. and Scherrer, L. C.,
Steroid hormone receptors. In Moudgil UK (ed.), Heat shock proteins and the cytoplasmic-nuclear trafficking of steroid receptors.
Birkhäuser, Boston, pp. 215-246 (1993).

DeFranco, D. B.,
Navigating steroid hormone receptors through the nuclear compartment.
Mol. Endocrinol.16(7), 1449-1455 (2002).

Picard, D.,
Posttranslational regulation of proteins by fusions to steroid binding domains.
Methods Enzymol. 194, 373 (1991).

Wang, Y. et al.,
Ligand-inducible and liver-specific target gene expression in transgenic mice.
Nat. Biotechnol.15, 239-243 (1996).

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