Construction of a targeting adenoviral vector carrying AFP promoter for expressing EGFP gene in AFP-producing hepatocarcinoma cell

AIM:To construct a recombinant adenoviral vector carryingAFPpromoter and EGFPgene for specific expression of EGFPgene in AFP producing hepatocellular carcinoma (HCC)HepG2 cells.METHODS:Based on the Adeno-X~(TM) expression system,thehuman immediate early cytomegalovirus promoter (P_(CMVIE))was removed from the plasmid,pshuttle,and replaced by a0.3 kb α-fetoprotein (AFP) promoter that was synthesizedby polymerase chain reaction (PCR).The enhanced greenfluorescent protein (EGFP) gene was inserted into the multi-clone site (MCS),and then the recombinant adenovirusvector carrying the 0.3 kb AFP promoter and EGFP genewas constructed.Cells of a normal liver cell line (LO2),ahepatocarcinoma cell line (HepG2) and a cervical cancercell line (HeLa) were transfectecl with the adenovirus.Northern blot and fluorescence microscopy were used todetect the expression of the EGFPgene at mRNA or proteinlevel in three different cell lines.RESULTS:The 0.3 kb AFP promoter was synthesizedthrough PCR from the human genome.The AFP promoterand EGFP gene were directly inserted into the plasmidpshuttle as confirmed by restriction digestion and DNAsequencing.Northern blot showed that EGFP gene wasmarkedly transcribed in HepG2 cells,but only slightly in LO2and HeLa cells.In addition,strong green fluorescence wasobserved in HepG2 cells under a fluorescence microscopy,but fluorescence was very weak in LO2 and HeLa cells.CONCLUSION:Under control of the 0.3 kb human AFPpromoter,the recombinant adenovirus vector carrying EGFPgene can be specially expressed in AFP-producing HepG2cells.Therefore,this adenovirus system can be used as anovel,potent and specific tool for gene-targeting therapyfor the AFP positive primary hepatocellular carcinoma.

Construction of a targeting adenoviral vector carrying AFP promoter for expressing EGFP gene in AFP-producing hepatocarcinoma cell

AIM: To construct a recombinant adenoviral vector carrying AFPpromoter and EGFPgene for specific expression of EGFP gene in AFP producing hepatocellular carcinoma (HCC) HepG2 cells.METHODS: Based on the Adeno-XTM expression system, the human immediate early cytomegalovirus promoter (PCMV IE) was removed from the plasmid, pshuttle, and replaced by a 0.3 kb α-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 EGFPgene 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 AFPpromoter 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.CONCLUSION: 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.