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AIM:To construct a gene modified hepatocellular carcinoma(HCC)specific EGFP expression vector regulated byabbreviated cis-acting element of AFP gene.METHODS:The minimal essential DNA segments of AFPgene enhancer and promoter were synthesized through PCRfrom Genome DNA of HepG2 cells.Gene fragments werethen cloned into the multiple cloning site of non-promoterEGFP vector pEGFP-1.Recombinant plasmid was transferredinto positive or negative AFP cell lines by means oflipofectamine.The expression of EGFP was tested byfluorescence microscope and flow cytometry.The effect ofall-trans retinoic acid(ATRA)on the expression of EGFP wastested in different concentrations.RESULTS:By the methods of restriction digestion andsequence analyses we confirmed that the length,positionand orientation of inserted genes of cis-acting element ofAFP were all correct.The transcription of EGFP was underthe control of AFP cis-acting element.The expressing EGFPcan only been detected in AFP producing hepatoma cells.The expression rate of EGFP in G418 screened cell line was34.9±4.1%.48 h after adding 1×10~(-7)M retinoic acid,EGFPexpression rate was 14.7±3.5 %.The activity of AFP genepromoter was significantly suppressed by addition of 1×10~(-7)Mretinoic acid(P<0.05,P=0.003,t=6.488).CONCLUSION:This recombinant expression vector can beused as a gene therapy vector for HCC.The expression oftumor killing gene will be confined within the site of tumorand the activity of which can be regulated by retinoic acid.
AIM: To construct a gene modified hepatocellular carcinoma (HCC) specific EGFP expression vector regulated by abbreviated cis-acting element of AFP gene. METHODS: The minimal essential DNA segments of AFP gene enhancer and promoter were synthesized by PCR from Genome DNA of HepG2 cells. Gene fragments werethen cloned into the multiple cloning site of non-promoter EGFP vector pEGFP-1.Recombinant plasmid was transferredinto positive or negative AFP cell lines by means oflipofectamine. The expression of EGFP was tested by fluorescence microscope and flow cytometry. The effect ofall-trans retinoic acid ( ATRA) on the expression of EGFP wastested in different concentrations .RESULTS: By the methods of restriction digestion and sequencing analyzes we confirmed that the length, positionand orientation of inserted genes of cis-acting element of AFP were all correct. The transcription of EGFP was undertaken of AFP cis-acting element. The expressed EGFPcan only been detected in AFP producing hepatoma cells.T The expression of EGFP in G418 screened cell line was 34.9 ± 4.1%. After 48 h after adding 1 × 10 -7 M retinoic acid, EGFP expression rate was 14.7 ± 3.5%. The activity of AFP gene promoter was significantly suppressed by addition of 1 × 10 -7 Mretinoic acid (P <0.05, P = 0.003, t = 6.488) .CONCLUSION: This recombinant expression vector can be used as a gene therapy vector for HCC.The expression of tumor killing gene will be confined within the site of tumorand the activity of which can be regulated by retinoic acid.