NF-kappa B related transgene expression in mouse tibial cranial muscle after pDNA injection followed or not by electrotransfer
Résumé
Background: When activated, NF-kappa B can promote the nuclear import and transcription of DNA possessing NF-kappa B consensus sequences. Here, we investigated whether NE-kappa B is involved in the plasmid electrotransfer process. Methods: Mouse tibial cranial muscles were transfected with plasmids encoding luciferase bearing or not NF-kappa B 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 I kappa B alpha were studied as a marker of NF-kappa B 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-kappa B 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 I kappa B alpha was slightly increased by simple pDNA injection and a little more by electrotransfer. We also observed a basal level of phosphorylated I kappa B alpha and thus of free NF-kappa B in the absence of any stimulation. General significance: pDNA electrotransfer can increase transgene expression independently of NF-kappa B. The insertion of NF-kappa B 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-kappa B in muscle might be sufficient to enhance the activity of pDNA bearing NF-kappa B consensus sequences. (C) 2014 Elsevier B.V. All rights reserved.