[Construction and identification of synchronous nitric oxide-amplified gene circuits controlled by radiation].
BACKGROUND
It has been proven that the positive feedback gene circuits can increase the expression level of interested genes, and the synchronization of genetic circuits can further enhance the efficacy of gene therapy. In order to obtain an enhanced and prolonged gene expression in target cells, a radiation controlled positive feedback genetic circuit is constructed via linking the c-fos promoter with the inducible nitric oxide synthase (iNOS) cDNA, which can be synchronized by nitric oxide (NO) intercellular messenger. Ultimately, the efficacy of radiogenetic therapy for cancer will be improved.


METHODS
Using the gene recombination techniques, the vector pfos-iNOS/green fluorescent protein (GFP) was generated by cloning the radiation-responsive c-fos promoter into the plasmid vector pIRES2-EGFP to replace the primary CMV promoter, and then inserting human iNOS cDNA downstream of c-fos promoter in the vector pIRES2-EGFP. The constructed plasmids were then downloaded into A549 cells with lipofectamine. With exposure of various doses of ionizing radiation, outputs of GFP and iNOS in the treated cells were observed and analyzed.


RESULTS
The interested plasmid was successfully constructed, proved by restriction enzyme digestion analysis. The outputs of GFP and iNOS in the transfected cells were markedly increased compared with the control cells after radiation, the peak level was seen in 16 hours after radiation.


CONCLUSIONS
A positive feedback genetic circuit is successfully developed, composed by c-fos promoter and iNOS cDNA, which can be synchronized by secreting the intercellular messenger NO. This genetic circuit will be utilized in further study.
Department of Oncology, Xinqiao Hospital, the Third Military Medical University, Chongqing 400037, P.R.China.
Baoguo
B
Kang
Weidong
W
Wang
Zhengtang
Z
Chen