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. 2003 Apr;9(4):688-91.
doi: 10.3748/wjg.v9.i4.688.

Construction of a regulable gene therapy vector targeting for hepatocellular carcinoma

Shao-Ying Lu  1 , Yan-Fang Sui, Zeng-Shan Li, Cheng-En Pan, Jing Ye, Wen-Yong Wang
Affiliations

Construction of a regulable gene therapy vector targeting for hepatocellular carcinoma

Shao-Ying Lu et al. World J Gastroenterol. 2003 Apr.

Abstract

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 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).

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 tumor and the activity of which can be regulated by retinoic acid.

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Figures

Figure 1
Figure 1
Gene map of pEGFP-1-EP.
Figure 2
Figure 2
PCR result of gene segments. A. 200 bp DNA marker; B. enhancer of AFP; C. promoter of AFP.
Figure 3
Figure 3
Enzyme digestion analysis of the recombinant ex-pression vector. A. 200 bp DNA marker; B. pEGFP-1-EP (Bgl II, Sal I, BamH I); C. pEGFP-1-EP (Xho I, BamH I); D. pEGFP-1-EP (Hind III); E. pEGFP-1(EcoR I, BamH I).
Figure 4
Figure 4
Transient expression of EGFP. A. EGFP expressing in HepG2; B. EGFP do not express in Hela cells.
Figure 5
Figure 5
The expression of EGFP suppressed by ATRA. A. stable expression of EGFP in HepG2; B. expression of EGFP suppressed by 2.5 × 10-7 M ATRA.
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