title: NF-kB related transgene expression in mouse tibial cranial muscle after pDNA injection followed or not by electrotransfer.
abstract: BACKGROUND
  When activated, NF-κB can promote the nuclear import and transcription of DNA possessing NF-κB consensus sequences. Here, we investigated whether NF-κB is involved in the plasmid electrotransfer process.
  
  
  METHODS
  Mouse tibial cranial muscles were transfected with plasmids encoding luciferase bearing or not NF-κB consensus sequences. Luciferase transgene expression was evaluated noninvasively by luminescence imaging and the number of pDNA copies in the same muscles by qPCR. RT-PCR of heat shock protein HsP70 mRNA evidenced cell stress. Western blots of phosphorylated IkBα were studied as a marker of NF-κB activation.
  
  
  RESULTS
  Intra-muscular injection of a plasmid bearing a weak TATA-like promoter results in a very low muscle transfection level. Electrotransfer significantly increased both the number of pDNA copy and the transgene expression of this plasmid per DNA copy. Insertion of NF-κB consensus sequences into pDNA significantly increased the level of gene expression both with and without electrotransfer. Electrotransfer-induced cellular stress was evidenced by increased HsP70 mRNA. Phosphorylated IκBα was slightly increased by simple pDNA injection and a little more by electrotransfer. We also observed a basal level of phosphorylated IκBα and thus of free NF-κB in the absence of any stimulation.
  
  
  CONCLUSIONS
  pDNA electrotransfer can increase transgene expression independently of NF-κB. The insertion of NF-κB consensus sequences into pDNA bearing a weak TATA-like promoter leads to enhanced transgene expression in muscle with or without gene electrotransfer. Finally, our results suggest that the basal amount of free NF-κB in muscle might be sufficient to enhance the activity of pDNA bearing NF-κB consensus sequences.
authors: U1022 INSERM, UMR8258 CNRS, Unité de Technologies Chimiques et Biologiques pour la Santé, Chimie ParisTech, Faculté de Pharmacie, 4 Avenue de l'Observatoire, 75006 Paris, France.
authors: S
authors: S
authors: Mahindhoratep
authors: U1022 INSERM, UMR8258 CNRS, Unité de Technologies Chimiques et Biologiques pour la Santé, Chimie ParisTech, Faculté de Pharmacie, 4 Avenue de l'Observatoire, 75006 Paris, France.
authors: H Ait
authors: HA
authors: Bouda
authors: U1022 INSERM, UMR8258 CNRS, Unité de Technologies Chimiques et Biologiques pour la Santé, Chimie ParisTech, Faculté de Pharmacie, 4 Avenue de l'Observatoire, 75006 Paris, France.
authors: Nelly
authors: N
authors: El Shafey
authors: U1022 INSERM, UMR8258 CNRS, Unité de Technologies Chimiques et Biologiques pour la Santé, Chimie ParisTech, Faculté de Pharmacie, 4 Avenue de l'Observatoire, 75006 Paris, France.
authors: D
authors: D
authors: Scherman
authors: V-SAT, UMR7199 CNRS, Université de Strasbourg, Faculté de Pharmacie, 67401 Illkirch Cedex, France.
authors: A
authors: A
authors: Kichler
authors: UPR4301 CNRS, rue Charles Sadron, 45071 Orléans Cedex 02, France.
authors: Ch
authors: Ch
authors: Pichon
authors: UPR4301 CNRS, rue Charles Sadron, 45071 Orléans Cedex 02, France.
authors: P
authors: P
authors: Midoux
authors: U1022 INSERM, UMR8258 CNRS, Unité de Technologies Chimiques et Biologiques pour la Santé, Chimie ParisTech, Faculté de Pharmacie, 4 Avenue de l'Observatoire, 75006 Paris, France.
authors: N
authors: N
authors: Mignet
authors: U1022 INSERM, UMR8258 CNRS, Unité de Technologies Chimiques et Biologiques pour la Santé, Chimie ParisTech, Faculté de Pharmacie, 4 Avenue de l'Observatoire, 75006 Paris, France.
authors: M F
authors: MF
authors: Bureau
keywords: Electroporation; IκBα; Muscle; NF-κB; Transcription; Transgene expression