A regulated, NFkappaB-assisted import of plasmid DNA into mammalian cell nuclei.
The success of synthetic DNA delivery systems in human gene therapy will be enhanced by increasing transfection efficiencies and by providing tighter control over targeting of the DNA into the nucleus. Here, we used DNA vectors that contain repetitive binding sites for the inducible transcription factor NFkappaB, which is transported into the nucleus by the nuclear import machinery. Nuclear entry of the modified vectors was augmented 12-fold and was associated with corresponding increase in gene expression. Depending on their position, the binding sites could also function as transcriptional enhancers, increasing gene expression levels up to an additional 19-fold. Notably, nuclear targeting of the DNA and transgene transcription could both be regulated by exogenous stimulators which modulate the intracellular distribution of NFkappaB. The approach provides a framework for the controlled targeting of constitutive or transcriptionally regulated synthetic vectors into mammalian cell nuclei.
Mesika
A
A
Department of Biological Chemistry, Weizmann Institute of Science, Rehovot 76100, Israel.
Grigoreva
I
I
Zohar
M
M
Reich
Z
Z
D000818:Animals; D001665:Binding Sites; D002467:Cell Nucleus / Q000378:metabolism*; D019084:Fluorescent Antibody Technique, Indirect; D018014:Gene Transfer Techniques; D015316:Genetic Therapy / Q000379:methods; D006367:HeLa Cells; D006801:Humans; D008156:Luciferases / Q000378:metabolism; D051379:Mice; D016328:NF-kappa B / Q000378:metabolism*; D019913:Nuclear Localization Signals; D010957:Plasmids / Q000378:metabolism*; D021381:Protein Transport; D013997:Time Factors; D014158:Transcription, Genetic; D014162:Transfection; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism
D016328:NF-kappa B; D019913:Nuclear Localization Signals; D014409:Tumor Necrosis Factor-alpha; D008156:Luciferases