Stringent rosiglitazone-dependent gene switch in muscle cells without effect on myogenic differentiation

Abstract

We have developed a gene switch based on the human transcription factor peroxisome proliferator-activated receptor gamma (PPARgamma) and its activation by rosiglitazone. However, ectopic expression of PPARgamma has been demonstrated to convert myogenic cells into adipocyte-like cells and, more generally, may interfere with the physiology of the target tissue. Consequently we modified the DNA-binding specificity of PPARgamma, resulting in a transcription factor that we named PPAR*. We demonstrated by histological and molecular assessment of cell phenotype that the overexpression of PPAR* did not alter the myogenic differentiation program of G8 myoblasts. We showed that PPAR* does not transactivate promoters containing PPARgamma-responsive elements but transactivates promoters containing PPAR*-responsive elements that are at least 80% identical to a 20-bp consensus. We improved the rosiglitazone-dependent gene switch by tuning PPAR* expression with a scaffold/matrix attachment region and by expressing both PPAR* and the reporter gene under the control of PPAR*-responsive elements. Treatment of cultured murine muscle cells (myotubes) with rosiglitazone induced reporter gene expression from assay background up to the level attained by a CMV I/E promoter-enhancer. These results indicate the potential of the PPAR* gene switch for use in gene therapy applications.

A novel mammalian expression system derived from components coordinating nicotine degradation in arthrobacter nicotinovorans pAO1

“…Currently available transcription control modalities capitalize on the generic design principle of the pioneering TET system: (i) clinically licensed tetracycline as inducer, (ii) bacterial repressor fused to mammalian cell-compatible transactivation domain as transactivator and (iii) transactivator-specific promoter assembled by fusing transactivator-specific operator modules to a minimal eukaryotic promoter. Besides their relationship to the TET configuration alternative transgene control systems differ significantly in the regulating small molecule (clinically licensed small-molecule drugs [antibiotics (7,9–11), immunosuppressive agents, (12), hormones and hormone agonists, (13–15), type-2 diabetes drug, (19,53), clinically inert compounds (17,18), temperature (16) and gaseous acetaldehyde (20)]), the origin of the transactivator [prokaryotic origin (7,9–11,16–18,20) (), mammalian origin (13,14,19,53)] and their promoter configurations [tandem operator modules, minimal promoter origin, relative promoter-operator spacing; see (40) as well as (52) for a non-limiting overview]. Despite the portfolio of different transgene regulation systems, there is nothing like the best control modality.…”

“…Following the generic design principle of the TET system, a wide variety of bacterial response regulators have been adapted for use as mammalian gene regulation systems, including those responsive to (i) tetracycline derivatives (7,8), (ii) streptogramin (9), (iii) macrolide (10) and (iv) coumermycin (11) antibiotics, (v) immunosuppressive rapamycin (12), hormones such as (vi) estrogen (13), (vii) progesterone (14) and (viii) ecdysone (15), (ix) temperature (16), (x) quorum-sensing molecules (17,18), (xi) the terpene cumate (), (xii) the type-2 diabetes drug roziglitazone (19) and (xiii) gaseous acetaldehyde (20). …”

Mammalian synthetic biology: emerging medical applications

“…Switches controlled by antibiotics [12,27,32], hormones and hormone analogues [29,40,95], quorum-sensing substances [53], and immune suppressive and anti-diabetic drugs [50,96] have recently been engineered expanding the arsenal of tools of synthetic biologists.…”

Pharmacologic transgene control systems for gene therapy

“…Reminiscent of the design of steroid hormone-inducible systems, a mutated peroxisome proliferator-activated receptor γ (PPAR * ) was constructed that exclusively heterodimerizes with the retinoic acid receptor RXR and activates artificial promoters harboring engineered PPAR * -responsive elements without interfering with native operators [66,67]. Similar to Hsp90-mediated sequestration of modified nuclear hormone receptors in the cytosol, Boutonnet and coworkers modified the mammalian translation-initiation factor 4G (eIF4G) by fusing it to a farnesylation signal of hRas (CAAX) so that recombinant eIF4G bound to cellular membranes [68,69].…”