The construction of bicistronic eukaryotic vector carrying green fluorescent protein and hytk gene and its expression in bladder carcinoma
- VernacularTitle:含绿色荧光蛋白和hytk双顺反子真核表达载体的构建及其在膀胱癌细胞中的表达
- Author:
Chuanzhong YE
;
Zhen LIN
;
Shiping CHEN
;
Fanglin ZHANG
;
Xuetao PEI
;
Liang LI
;
Ka FENG
- Publication Type:Journal Article
- Keywords:
Fluorescence;
Genes;
Ribosome;
Bladder neoplasms
- From:
Chinese Journal of Pathophysiology
1999;0(09):-
- CountryChina
- Language:Chinese
-
Abstract:
AIM: To facilitate the suicide gene delivery into neoplasm, a chimeric gene of HSV-tk and green fluorescent protein (gfp) was constructed. METHODS: Molecular cloning technique was used to construct this kind of eukaryotic vector. The internal ribosome entry site (IRES) of encephalomyocarditis virus (EMCV), which could coordinate expression of two genes in a single vector, was optioned. By using liposome-mediated transfection, eukaryotic expression vector tgCMV/hytk-IRES-gfp was transfected into human bladder carcinoma cells EJ. RESULTS: A bicistronic eukaryotic vector carrying gfp and hygromycin phosphotransferase-thymidine kinase fusion (hytk) gene was constructed. The results of PCR and microscopy detection show that the hytk-IRES-gfp gene was successfully transferred into EJ cells. There were no differences in the growth pattern or the morphology between EJ and EJ/hytk-GFP cells. In vitro experiments demonstrated dose- and time-dependent cell killing by transduction of the hytk-IRES-gfp gene followed by GCV treatment. The IC50 (the concentration required to elicit 50% growth inhibition) was 2.16 mg/L in treatment with GCV for 72 hours. CONCLUSION: These results suggest that this new kind of eukaryotic vector could serves as a new tool and method for neoplasm gene therapy.
