Construction of a targeting adenoviral vector carrying AFP promoter for expressing EGFP gene in AFP-producing hepatocarcinoma cell.
OBJECTIVE
To construct a recombinant adenoviral vector carrying AFP promoter and EGFP gene for specific expression of EGFP gene in AFP producing hepatocellular carcinoma (HCC) HepG2 cells.


METHODS
Based on the Adeno-X expression system, the human immediate early cytomegalovirus promoter (PCMV IE) was removed from the plasmid, pshuttle, and replaced by a 0.3 kb alpha-fetoprotein (AFP) promoter that was synthesized by polymerase chain reaction (PCR). The enhanced green fluorescent protein (EGFP) gene was inserted into the multi-clone site (MCS), and then the recombinant adenovirus vector carrying the 0.3 kb AFP promoter and EGFP gene was constructed. Cells of a normal liver cell line (LO2), a hepatocarcinoma cell line (HepG2) and a cervical cancer cell line (HeLa) were transfected with the adenovirus. Northern blot and fluorescence microscopy were used to detect the expression of the EGFP gene at mRNA or protein level in three different cell lines.


RESULTS
The 0.3 kb AFP promoter was synthesized through PCR from the human genome. The AFP promoter and EGFP gene were directly inserted into the plasmid pshuttle as confirmed by restriction digestion and DNA sequencing. Northern blot showed that EGFP gene was markedly transcribed in HepG2 cells, but only slightly in LO2 and HeLa cells. In addition, strong green fluorescence was observed in HepG2 cells under a fluorescence microscopy, but fluorescence was very weak in LO2 and HeLa cells.


CONCLUSIONS
Under control of the 0.3 kb human AFP promoter, the recombinant adenovirus vector carrying EGFP gene can be specially expressed in AFP-producing HepG2 cells. Therefore, this adenovirus system can be used as a novel, potent and specific tool for gene-targeting therapy for the AFP positive primary hepatocellular carcinoma.
Shi
Yu-Jun
YJ
Department of General Surgery, the Second College of Clinical Medicine and the Second Affiliated Hospital of Chongqing University of Medical Sciences, Chongqing 400010, China.
Gong
Jian-Ping
JP
Liu
Chang-An
CA
Li
Xu-Hong
XH
Mei
Ying
Y
Mi
Can
C
Huo
Yan-Ying
YY
D000256:Adenoviridae / Q000235:genetics*; D001483:Base Sequence; D015316:Genetic Therapy / Q000379:methods; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins; D006367:HeLa Cells; D006801:Humans; D007202:Indicators and Reagents / Q000378:metabolism; D008113:Liver Neoplasms; D008164:Luminescent Proteins / Q000235:genetics; D008969:Molecular Sequence Data; D010957:Plasmids; D011401:Promoter Regions, Genetic; D000509:alpha-Fetoproteins / Q000235:genetics*
D007202:Indicators and Reagents; D008164:Luminescent Proteins; D000509:alpha-Fetoproteins; D049452:Green Fluorescent Proteins