title: [The construction of IGF-II P4 promoter-driven tk expression vector].
abstract: OBJECTIVE
  To investigate the selective killing effect of herpes simplex virus-thymidine kinase/ganciclovir (HSV-tk/GCV) suicide gene system controlled by human IGF-II P4 promoter on HCC cells in vitro.
  
  
  METHODS
  Recombinant shuttle plasmid vectors driven by IGF-II P4 promoter and driven by CMV promoter were constructed by techniques of gene recombination. The recombinant shuttle plasmids were then transfected into HepG2 and HeLa cells by techniques of lipidosome transfection. EGFP expression was detected by fluoroscopy. Tk and EGFP mRNA expression were detected by RT-PCR. The selective killing effect after GCV application was determined with MTT method. Statistical analysis was performed with ANOVA analysis.
  
  
  RESULTS
  Identification of pDC316-tkEGFP-P4 by enzyme digestion and sequencing analysis showed that the construction of the recombinant shuttle plasmid was correct. It was found that green fluorescence protein could only be seen in HepG2 cells, not in HeLa cells. The results of RT-PCR showed only two bands could be seen in the samples of pDC316-tkEGFP-P4 transfected HepG2 cells. The growth of HepG2 cells transfected with pDC316-tkEGFP-CMV and pDC316-tkEGFP-P4 were inhibited remarkably, the growth inhibition rates were 6.95% +/- 0.67%, 24.99% +/- 1.53%, 49.68% +/- 1.68%, 71.85% +/- 3.28% and 4.83% +/- 0.35% vs 17.34% +/- 1.15%, 30.17% +/- 1.30%, 40.39% +/- 0.82% (F = 24.055, P < 0.05), respectively. The growth of HeLa cells transfected with pDC316-tkEGFP-CMV were also inhibited, the growth inhibition rates were 6.36% +/- 0.83%, 23.95% +/- 1.72%, 45.13% +/- 1.64% and 69.38% +/- 3.17%, respectively. The growth of HeLa cells transfected with pDC316-tkEGFP-P4 was not inhibited. The growth inhibition rates were 0.91 +/- 0.04, 1.18 +/- 1.32, 1.19 +/- 0.10 and 1.32 +/- 0.05 (F = 26.469, P < 0.01) , respectively.
  
  
  CONCLUSIONS
  The shuttle plasmid vector carrying the tkEGFP fusion protein gene driven by IGF-II P4 promoter has been constructed successfully and its specific expression in HepG2 cells provided the basis for targeted gene therapy for HCC.
authors: Department of Gastroenterology, First Affiliated Hospital, Jinan University, Guangzhou 510630, China.
authors: Hong-Ke
authors: HK
authors: Zhou
authors: Dong-Hua
authors: DH
authors: Yang
authors: Shao-Hui
authors: SH
authors: Tang
authors: Wei
authors: W
authors: Huang
mesh_terms: D045744:Cell Line, Tumor; D041722:Genes, Transgenic, Suicide / Q000235:genetics*; D005822:Genetic Vectors; D006801:Humans; D007335:Insulin-Like Growth Factor II / Q000235:genetics* / Q000494:pharmacology; D010957:Plasmids; D011401:Promoter Regions, Genetic; D013937:Thymidine Kinase / Q000235:genetics*; D014162:Transfection
chemical_list: D007335:Insulin-Like Growth Factor II; D013937:Thymidine Kinase