Background:
Although lipofection-induced tnf-alpha can activate nuclear factor kappab (nf-kappab), which, in turn, increases the transgene expression from plasmid dna in which any nf-kappab responsive element is incorporated, no attempts have been made to use such biological responses as nf-kappab activation against a vector to enhance vector-mediated gene transfer.
Methods:
A lipoplex composed of n-[1-(2,3-dioleyloxy)propyl]-n,n,n-trimethylammonium and cholesterol liposome and plasmid dna encoding firefly luciferase under the control of the cytomegalovirus immediate early promoter (pcmv-luc) was intravenously injected into mice. luciferase activity as well as nf-kappab activation in the lung were evaluated. then, a novel plasmid dna, pcmv-kappab-luc, was constructed by inserting 5 repeats of nf-kappab-binding sequences into the pcmv-luc.
Results:
Nf-kappab in the lung was activated by injection of the lipoplex and its nuclear localization was observed. an injection of lipopolysaccharide 30 min prior to the lipofection further activated nf-kappab. at the same time, the treatment significantly increased the transgene expression by lipoplex, suggesting a positive correlation between expression and nf-kappab activity. based on these findings, we tried to enhance the lipoplex-based transgene expression by using nf-kappab activation. the lipoplex consisting of pcmv-kappab-luc showed a 4.7-fold increase in transgene expression in the lung compared with that with pcmv-luc.
Conclusions:
We demonstrated that nf-kappab activation by lipoplex can be used to enhance lipoplex-mediated transgene expression by inserting nf-kappab-binding sequences into plasmid dna. these findings offer a novel method for designing a vector for gene transfer in conjunction with biological responses to it.