The Expression of Adenoviral Mediated gfp Reporter Gene in Tumor Cells Induced and Regulated by Irradiation.
In order to establish a radiation inducible gene expression system for cancer gene therapy, the promoter sequence of radiation-inducible Egr-1 gene was amplified from genomic DNA of BALB/c mouse with PCR method, and linked to gfp reporter gene. Then the pEgr-gfp expression cassette was subcloned into an adenoviral shuttle plasmid to generate recombinant adenovirus of AdEgr-GFP by using a novel, high efficient method of homologous recombination in bacteria. After infection with AdEgr-GFP, MM45T.Li tumor cells were exposed to different doses of gamma-irradiation from 0 Gy to 15 Gy in vitro. The percentage of GFP expression positive cells increased greatly in a dose-dependent manner as detected by FACS and Western blot analysis. For in vivo study, AdEgr-GFP were injected intratumorally, and tumor site received different doses of local gamma-irradiation 48 h after injection, and after 8 h the tumor samples were biopsed for investigating the GFP expression. Tumor tissue image analysis revealed that gamma-irradiation could markedly increase GFP expression in a dose-dependent manner as compared with that of non-irradiated control group. Our results indicate that the irradiation can effectively control adenoviral-mediated GFP expression in tumor cells via Egr-1 promoter, and these data laid basis for further gene radiotherapy study.
Wei
Dao-Yan
DY
Department of Biochemistry, Research Center for Human Gene Therapy, Shanghai Second Medical University, Shanghai 200025, China. sschen@shsmu.edu.cn
Dai
Bing-Bing
BB
Chen
Shi-Shu
SS
Wang
Zhong-He
ZH