The piggyBac transposon is an integrating non-viral gene transfer vector that enhances the efficiency of GDEPT.
Gene-directed enzyme prodrug therapy (GDEPT) is a strategy developed to selectively target cancer cells. However, the clinical benefit is limited due to its poor gene transfer efficiency. To overcome this obstacle, we took advantage of piggyBac (PB) transposon, a natural non-viral gene vector that can induce stable chromosomal integration and persistent gene expression in vertebrate cells, including human cells. To determine whether the vector can also mediate stable gene expression in ovarian cancer cells, we constructed a PB transposon system that simultaneously expresses the Herpes simplex virus thymidine kinase (HSV-tk) gene and the monomeric red fluorescent protein (mRFP1) reporter gene. The recombinant plasmid, pPB/TK, was transfected into ovarian adenocarcinoma cells SKOV3 with FuGENE HD reagent, and the efficiency was given by the percentage of mRFP1-positive cells detected by flow cytometry and confocal microscopy. The specific expression of HSV-tk in transfected cells was confirmed by RT-PCR and western blotting. The sensitivity of transfected cells to pro-drug ganciclovir (GCV) was determined by methylthiazoletetrazolium (MTT) assay. A total of 56.4 +/- 8.4% cells transfected with pPB/TK were mRFP1 positive, compared to no measurable mRFP1 expression in pORF-HSVtk-transfected cells. The expression level of HSV-tk in pPB/TK-transfected cells was 10 times higher than in pORF-HSVtk-transfected cells. The results show that pPB/TK transfection increases the sensitivity of cells to GCV in a dose-dependent manner. Our data indicate that the PB transposon system could enhance the anti-tumor efficiency of GDEPT in ovarian cancer.
Obstetrics and Gynecology Hospital, Fudan University, Shanghai, China.
Yu
Y
Kang
Xiao-Yan
XY
Zhang
Wei
W
Jiang
Chao-Qun
CQ
Wu
Chun-Mei
CM
Chen
Jian-Ren
JR
Gu
Yu-Fang
YF
Zheng
Cong-Jian
CJ
Xu
D000230:Adenocarcinoma / Q000628:therapy*; D045744:Cell Line, Tumor; D004251:DNA Transposable Elements / Q000235:genetics*; D019008:Drug Resistance, Neoplasm; D005260:Female; D015774:Ganciclovir / Q000494:pharmacology; D015316:Genetic Therapy / Q000379:methods*; D005822:Genetic Vectors / Q000235:genetics*; D006801:Humans; D008164:Luminescent Proteins / Q000235:genetics; D010051:Ovarian Neoplasms / Q000628:therapy*; D011355:Prodrugs / Q000494:pharmacology*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D013937:Thymidine Kinase / Q000235:genetics; D014162:Transfection; D000097573:Red Fluorescent Protein
D004251:DNA Transposable Elements; D015774:Ganciclovir; D008164:Luminescent Proteins; D011355:Prodrugs; D011993:Recombinant Fusion Proteins; D013937:Thymidine Kinase; D000097573:Red Fluorescent Protein