Mahindhoratep S, Bouda H Ait, El Shafey Nelly, Scherman D, Kichler A, Pichon Ch, Midoux P, Mignet N, Bureau M F
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.
V-SAT, UMR7199 CNRS, Université de Strasbourg, Faculté de Pharmacie, 67401 Illkirch Cedex, France.
Biochim Biophys Acta. 2014 Nov;1840(11):3257-63. doi: 10.1016/j.bbagen.2014.06.013. Epub 2014 Jun 26.
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.
激活后，核因子κB（NF-κB）可促进具有NF-κB共有序列的DNA的核输入和转录。在此，我们研究了NF-κB是否参与质粒电转染过程。
用编码带有或不带有NF-κB共有序列的荧光素酶的质粒转染小鼠胫前肌。通过发光成像非侵入性地评估荧光素酶转基因表达，并通过qPCR评估同一肌肉中pDNA拷贝数。热休克蛋白HsP70 mRNA的RT-PCR证明细胞应激。研究磷酸化IκBα的蛋白质印迹作为NF-κB激活的标志物。
肌内注射带有弱TATA样启动子的质粒导致肌肉转染水平非常低。电转染显著增加了每个DNA拷贝的pDNA拷贝数和该质粒的转基因表达。将NF-κB共有序列插入pDNA中，无论有无电转染，均显著提高了基因表达水平。电转染诱导的细胞应激通过增加的HsP70 mRNA得以证明。简单注射pDNA可使磷酸化IκBα略有增加，电转染使其增加更多。我们还观察到在没有任何刺激的情况下，磷酸化IκBα以及游离NF-κB的基础水平。
pDNA电转染可独立于NF-κB增加转基因表达。将NF-κB共有序列插入带有弱TATA样启动子的pDNA中，无论有无基因电转染，均可导致肌肉中转基因表达增强。最后，我们的结果表明，肌肉中游离NF-κB的基础量可能足以增强带有NF-κB共有序列的pDNA的活性。
