Stringent rosiglitazone-dependent gene switch in muscle cells without effect on myogenic differentiation.
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.
Tascou
Semi
S
Gencell S.A.S., 72-82 Rue Léon Geoffroy, 94408 Vitry sur Seine, France.
Sorensen
Tine-Kring
TK
Glénat
Valérie
V
Wang
Manping
M
Lakich
Mélissa M
MM
Darteil
Raphaël
R
Vigne
Emmanuelle
E
Thuillier
Vincent
V
D019943:Amino Acid Substitution; D000818:Animals; D002454:Cell Differentiation; D002460:Cell Line; D004268:DNA-Binding Proteins / Q000378:metabolism; D005786:Gene Expression Regulation; D017930:Genes, Reporter / Q000235:genetics; D005822:Genetic Vectors / Q000235:genetics; D008156:Luciferases / Q000032:analysis / Q000235:genetics; D051379:Mice; D018485:Muscle Fibers, Skeletal / Q000166:cytology / Q000378:metabolism*; D009154:Mutation; D032446:Myoblasts / Q000378:metabolism; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D011965:Receptors, Glucocorticoid / Q000235:genetics; D020218:Response Elements; D000077154:Rosiglitazone; D045162:Thiazolidinediones / Q000494:pharmacology*; D014162:Transfection
D004268:DNA-Binding Proteins; D011965:Receptors, Glucocorticoid; D045162:Thiazolidinediones; D000077154:Rosiglitazone; D008156:Luciferases