[Construction of recombinant adenoviruses encoding TK suicide gene driven by VEGF promoter using efficient AdEasier-1 system].
OBJECTIVE
One of the bottlenecks of suicide gene therapy is the low specific expression of suicide genes in tumor cells. Using tumor specific promoter to modulate suicide genes resulting in high specific expression of genes in tumor cells has became a hot research topic of tumor suicide gene therapy. It has showed that vascular endothelial growth factor (VEGF) over-expresses in almost all solid tumors, but not in normal tissues, which is highly relevant to the up-regulation of VEGF promoter (VEGFP) activity. This study was to use the simplified and efficient AdEasier-1 system to generate recombinant adenoviruses encoding TK gene driven by VEGFP, and determine whether VEGFP could increase the expression of TK suicide gene in tumor cells.


METHODS
A fragment containing VEGFP was isolated from pEGFP-1-SV-VEGFP (Not I /Xho I), and cloned into the shuttle plasmid pAdTrack resulting in pAdTrack-VEGFP. TK gene by polyadenylation site was cut from pREP8-TK by HindIII,and XbaI, and subcloned into pAdTrack-VEGFP resulting in pAdtrack-VEGFP-TK, which was linearized by PmeI and transformed into AdEasier-1 Cells. Transformants were selected on LB agar plates containing 25 microg/mL kanamycin, and positive pAdEasy-VEGFP-TK was identified by electrophoretic analysis and enzymatic digestion, then digested with PacI, and transfected into 293 cells to produce the recombinant adenovirus Ad-VEGFP-TK. Ad-VEGFP-TK was finally confirmed by polymerase chain reaction (PCR) procedure, and DNA sequence analysis.


RESULTS
The pAdTrack-VEGFP-TK (about 12 kb)and pAdEasy-VEGFP-TK(larger than 33 kb), both selectable by kanamycin resistance, could be clearly identified by electrophoretic analysis. Digesting pAdEasy-VEGFP-TK with PacI resulted in 1 specific small fragment (about 3.0 kb), and 1 large fragment (larger than 33 kb). The pAdEasy-VEGFP-TK was successfully constructed at a frequency of 60% (6/10). After being packaged in 293 cells, and purified by CsCl banding, the recombinant adenovirus Ad-VEGFP-TK, whose titer was as high as 5.6x1012 viral particle/ml, were produced, which were further proved to be correct by enzyme digestion, PCR analysis, and DNA sequence analysis.


CONCLUSIONS
AdEasier-1 system is an efficient way to construct the recombinant adenoviruses encoding TK gene under control of VEGFP.
Chen
Jian-Fa
JF
Department of General Surgery, Zhujiang Hospital, The First Military Medical University, Guangzhou, Guangdong, 510282, PR China. cjf.fimmu@eyou.com
Huang
Zong-Hai
ZH
Huang
Yuan-Yuan
YY
Song
Hui-Juan
HJ
Che
Xiao-Yan
XY
D000256:Adenoviridae / Q000235:genetics*; D045744:Cell Line, Tumor; D041722:Genes, Transgenic, Suicide; D015316:Genetic Therapy; D006801:Humans; D007668:Kidney / Q000166:cytology / Q000378:metabolism; D009369:Neoplasms / Q000628:therapy; D011401:Promoter Regions, Genetic; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D011995:Recombination, Genetic; D013937:Thymidine Kinase / Q000235:genetics*; D014162:Transfection; D042461:Vascular Endothelial Growth Factor A / Q000235:genetics*
D011994:Recombinant Proteins; D042461:Vascular Endothelial Growth Factor A; D013937:Thymidine Kinase