The Expression of
Adenoviral Mediated gfp
Reporter Gene in Tumor Cells Induced and Regulated by Irradiation
WEI Dao-Yan, DAI Bing-Bing, CHEN Shi-Shu*
( Department of Biochemistry, Research Center for Human Gene Therapy,
Shanghai Second Medical University, Shanghai 200025, China )
WANG Zhong-He
( Department of Radiology, Shanghai Ninth Peoples Hospital, Shanghai 200011,
China )
Abstract 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 γ-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 γ-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 γ-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.
Key words regulation of gene expression；irradiation；Erg-1 promoter；gfp reporter gene； recombinant adenovirus
*Corresponding author：Tel,86-21-63846590-452；Fax,86-21-63842916；e-mail, [email protected]