Inhibition of HBs-GFP fusion gene expression by RNA interference.
- Author:
Zheng-gang YANG
1
;
Zhi CHEN
;
Qin NI
;
Xiu-cheng PAN
;
Han-ying JIN
;
Xing-yi LI
Author Information
- Publication Type:Journal Article
- MeSH: Carcinoma, Hepatocellular; pathology; Gene Expression Regulation, Viral; Green Fluorescent Proteins; genetics; Hepatitis B Surface Antigens; genetics; Hepatitis B virus; genetics; Humans; Liver Neoplasms; pathology; RNA Interference; RNA, Small Interfering; Recombinant Fusion Proteins; biosynthesis; genetics; Transfection; Tumor Cells, Cultured
- From: Journal of Zhejiang University. Medical sciences 2005;34(2):110-115
- CountryChina
- Language:Chinese
-
Abstract:
OBJECTIVETo develop an effective report gene system to test the effect of small interfering RNA (siRNA).
METHODSHBV S gene was fused with enhanced green fluorescent protein (EGFP) gene to form HBs-GFP and the plasmid containing HBs-GFP was constructed. A vector expressing small hairpin RNA (shRNA) pAVU6 + 4sh357 was also constructed. Two plasmids were co-transfected into HepG2 cells transiently. The fluorescence of HBs-GFP was detected by fluorescence-activated cell sorting (FACS). The mRNA expression in HepG2 cells was detected by conventional RT-PCR and real-time PCR.
RESULTSsiRNA inhibited the expression of HBs-GFP 72 hours post transfection. The fluorescence of HBs-GFP in HepG2 cells treated with pAVU6+4sh357 was reduced by 55.4% compared with that of controls. The HBs-GFP expression in HepG2 cells treated with pAVU6+4sh357 was reduced by 76.3% and 90% as measured with conventional RT-PCR and real-time PCR, respectively.
CONCLUSIONThis investigation demonstrated siRNA derived from shRNA expression vectors can inhibit the expression of HBs-GFP in HepG2 cells.
