[Construction of recombinant adenoviruses containing cytosine deaminase gene driven by the vascular endothelial growth factor promoter using an AdEasier-1 system].
OBJECTIVE
To efficiently construct a recombinant adenovirus containing cytosine deaminase (CD) gene driven by vascular endothelial growth factor (VEGF) promoter using an AdEasier-1 system and observe its killing effect on LoVo cells in vitro.


METHODS
The CD gene was amplified by PCR, and amplicons were cloned in JM109 bacteria, and then recombined into pREP8 to obtain pREP8-CD, which was then digested by HindIIIand XbaIfor the CD fragment with polyadenylation site (CD-pA) subcloned into the VEGFP -containing shuttle plasmid pAdtrack-VEGFP to generate pAdtrack-VEGFP-CD-pA. After linearization with PmeI, pAdtrack-VEGFP-CD-pA was transformed into AdEasier-1 cells, and the transformants were selected on LB agar plates containing 25 microg/ml kanamycin followed by identification of the positive pAdEasy-VEGFP-CD with electrophoretic analysis and enzymatic digestion. pAdEasy-VEGFP-CD was then digested with PacIand transfected into 293 cells to produce the recombinant adenovirus Ad-VEGFP-CD, which was finally confirmed by PCR. The positive recombinant adenoviruses were transfected into LoVo cells to observe their in vitro anti-tumor effect.


RESULTS
pAdEasy-VEGFP-CD was constructed with a success rate of 70%. After being packaged in 293 cells and purified by CsCl banding, the titer of the recombinant adenovirus Ad-VEGFP-CD reached as high as 4.8x10(12) CFU particle/ml, and the adenovirus was further confirmed by PCR analysis. In the presence of the prodrug 5-FC, the recombinant adenoviruses remarkably inhibited the growth of LoVo cells.


CONCLUSIONS
The recombinant adenoviruses containing CD gene under the control of VEGF promoter can be efficiently generated using the AdEasier-1 system, and exhibit potent anti-tumor effect in vitro.
Chen
Jian-fa
JF
Department of General Surgery, Zhujiang Hospital, First Military Medical University, Guangzhou 510282, China. cjf.fimmu@eyou.com
Hunag
Zong-hai
ZH
Yu
Jin-long
JL
Li
Zhou
Z
Che
Xiao-yan
XY
D000256:Adenoviridae / Q000235:genetics*; D043525:Cytosine Deaminase / Q000235:genetics*; D015316:Genetic Therapy; D006801:Humans; D009369:Neoplasms / Q000628:therapy*; D010957:Plasmids; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic; D011995:Recombination, Genetic; D042461:Vascular Endothelial Growth Factor A / Q000235:genetics*
D042461:Vascular Endothelial Growth Factor A; D043525:Cytosine Deaminase