Transcriptional activation of the metallothionein I gene by electric pulses in vivo: basis for the development of a new gene switch system.
BACKGROUND
In vivo gene transfer to skeletal muscle is a promising strategy for the treatment of muscular disorders and for the systemic delivery of therapeutic proteins. Nevertheless, for a safe and effective protein production, the spatial and temporal control of gene expression is critical. The existing regulating systems rely on the use of an exogenously regulatory protein and/or an inducer drug whose pharmacological properties are of major concerns for therapeutic applications in humans. Therefore, new strategies based on endogenous regulatable elements have been explored.


METHODS
Gene expression profiles of skeletal muscle submitted or not to electrical pulses and harvested at different times were compared using the Affymetrix GeneChip technology. The endogenous metallothionein promoter was studied by Northern blot and semiquantitative and quantitative RT-PCR. The inducibility of the metallothionein I promoter placed in a plasmid exogenous context was studied using the murine SEAP reporter gene.


RESULTS
The expression of metallothionein I mRNA is significantly increased 6 h after electric pulses delivery. This induction is transient. Identical MT-I expression level is observed after several sequential series of pulses delivery. We demonstrated as well that the MT-II promoter was sensitive to electric pulses delivery. Moreover, the metallothionein I promoter, placed in a plasmid context in front of a reporter gene, was also activated by the application of transient electric field.


CONCLUSIONS
We identified a promoter highly inducible by the controlled electric stimuli applied for electrotransfer experiments. The use of the metallothionein promoter is promising for the time-control by physical stimuli of the expression of a therapeutic gene.
Rubenstrunk
Anne
A
UMR7001 CNRS/ENSCP/Gencell SA, Vectorologie Moléculaire et Cellulaire, Centre de Recherche de Vitry-Alfortville, 13 quai Jules Guesde, BP 14, 94403 Vitry Sur Seine, France.
Orsini
Cécile
C
Mahfoudi
Abderrahim
A
Scherman
Daniel
D
D000818:Animals; D002135:Calcium-Binding Proteins / Q000008:administration & dosage; D018274:Electroporation / Q000379:methods*; D005786:Gene Expression Regulation; D005822:Genetic Vectors; D007273:Injections, Intramuscular; D008668:Metallothionein / Q000235:genetics* / Q000378:metabolism; D051379:Mice; D018482:Muscle, Skeletal / Q000378:metabolism; D010957:Plasmids / Q000235:genetics; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000378:metabolism; D015533:Transcriptional Activation
D002135:Calcium-Binding Proteins; D012333:RNA, Messenger; C025673:calciphorin; D008668:Metallothionein