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Mahindhoratep, S., Bouda, H. A., El Shafey, N., Scherman, D., Kichler, A., Pichon, C., Midoux, P., Mignet, N. and Bureau, M. F.

NF-kB related transgene expression in mouse tibial cranial muscle after pDNA injection followed or not by electrotransfer

Biochim Biophys Acta 1840 (11) 3257-3263 - doi : 10.1016/j.bbagen.2014.06.013

par Frapart - publié le , mis à jour le

Abstract :

BACKGROUND : When activated, NF-kappaB can promote the nuclear import and transcription of DNA possessing NF-kappaB consensus sequences. Here, we investigated whether NF-kappaB is involved in the plasmid electrotransfer process. METHODS : Mouse tibial cranial muscles were transfected with plasmids encoding luciferase bearing or not NF-kappaB 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 IkBalpha were studied as a marker of NF-kappaB 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-kappaB 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 IkappaBalpha was slightly increased by simple pDNA injection and a little more by electrotransfer. We also observed a basal level of phosphorylated IkappaBalpha and thus of free NF-kappaB in the absence of any stimulation. GENERAL SIGNIFICANCE : pDNA electrotransfer can increase transgene expression independently of NF-kappaB. The insertion of NF-kappaB 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-kappaB in muscle might be sufficient to enhance the activity of pDNA bearing NF-kappaB consensus sequences.