[Inhibition of HBs-GFP fusion gene expression by RNA interference].
To develop an effective report gene system to test the effect of small interfering RNA (siRNA).



HBV 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.



siRNA 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.



This investigation demonstrated siRNA derived from shRNA expression vectors can inhibit the expression of HBs-GFP in HepG2 cells.
Yang
Zheng-gang
ZG
Institute of Infectious Diseases, The First Affiliated Hospital, College of Medicine, Zhejiang University, Hangzhou 310003, China.
Chen
Zhi
Z
Ni
Qin
Q
Pan
Xiu-cheng
XC
Jin
Han-ying
HY
Li
Xing-yi
XY
D006528:Carcinoma, Hepatocellular / Q000473:pathology; D015967:Gene Expression Regulation, Viral; D049452:Green Fluorescent Proteins / Q000235:genetics*; D006514:Hepatitis B Surface Antigens / Q000235:genetics*; D006515:Hepatitis B virus / Q000235:genetics; D006801:Humans; D008113:Liver Neoplasms / Q000473:pathology; D034622:RNA Interference; D034741:RNA, Small Interfering; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics; D014162:Transfection; D014407:Tumor Cells, Cultured
D006514:Hepatitis B Surface Antigens; D034741:RNA, Small Interfering; D011993:Recombinant Fusion Proteins; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins