Construction of a regulable gene therapy vector targeting for hepatocellular carcinoma.
OBJECTIVE
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 through PCR from Genome DNA of HepG2 cells. Gene fragments were then cloned into the multiple cloning site of non-promoter EGFP vector pEGFP-1. Recombinant plasmid was transferred into positive or negative AFP cell lines by means of lipofectamine. The expression of EGFP was tested by fluorescence microscope and flow cytometry. The effect of all-trans retinoic acid (ATRA) on the expression of EGFP was tested in different concentrations.


RESULTS
By the methods of restriction digestion and sequence analyses we confirmed that the length, position and orientation of inserted genes of cis-acting element of AFP were all correct. The transcription of EGFP was under the control of AFP cis-acting element. The expressing EGFP can only been detected in AFP producing hepatoma cells. The expression rate of EGFP in G418 screened cell line was 34.9+/-4.1 %. 48 h after adding 1X10(-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 x 10(-7)M retinoic acid (P<0.05, P=0.003, t=6.488).


CONCLUSIONS
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 tumor and the activity of which can be regulated by retinoic acid.
Lu
Shao-Ying
SY
Department of Pathology, Fourth Military Medical University, Xi'an 710032, China.
Sui
Yan-Fang
YF
Li
Zeng-Shan
ZS
Pan
Cheng-En
CE
Ye
Jing
J
Wang
Wen-Yong
WY
D006528:Carcinoma, Hepatocellular / Q000235:genetics* / Q000628:therapy; D004742:Enhancer Elements, Genetic; D005819:Genetic Markers; D015316:Genetic Therapy / Q000379:methods*; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins; D006801:Humans; D008113:Liver Neoplasms / Q000235:genetics* / Q000628:therapy; D008164:Luminescent Proteins / Q000235:genetics; D011401:Promoter Regions, Genetic; D011993:Recombinant Fusion Proteins / Q000032:analysis; D015183:Restriction Mapping; D014407:Tumor Cells, Cultured; D000509:alpha-Fetoproteins / Q000235:genetics*
D005819:Genetic Markers; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D000509:alpha-Fetoproteins; D049452:Green Fluorescent Proteins