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

Abstract : 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. 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. 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. 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.
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Journal articles
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https://hal.archives-ouvertes.fr/hal-01179311
Contributor : Isabelle Frapart <>
Submitted on : Wednesday, July 22, 2015 - 11:12:52 AM
Last modification on : Thursday, June 20, 2019 - 12:22:09 PM

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  • HAL Id : hal-01179311, version 1
  • PUBMED : 24973564

Citation

S Mahindhoratep, H Ait Bouda, Nelly El Shafey, D Scherman, A Kichler, et al.. NF-kB related transgene expression in mouse tibial cranial muscle after pDNA injection followed or not by electrotransfer.. BBA - Biochimica et Biophysica Acta, Elsevier, 2014, 1840 (11), pp.3257-3263. ⟨hal-01179311⟩

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