[Highly efficient construction of recombinant adenovirus containing double suicide gene driven by cytomegalovirus promoter using two-step CaCl2 transformation method].
OBJECTIVE
To construct recombinant adenovirus containing double suicide gene driven by cytomegalovirus (CMV) promoter.


METHODS
CD and TK gene were amplified by PCR and cloned into the shuttle vector pAdtrack CMV, and the resultant plasmid pAdtrack CMV-CDTK was linearized with PmeI and transformed into competent AdEasier-1 cells prepared by CaCl2 method. PacI digestion of identified recombinant plasmid DNA was performed before it was transected into 293 cells to package adenovirus, which was used to infect endothelial cells in vitro after adenovirus titer determination.


RESULTS
Chemical transformation of linearized transfer plasmid into AdEasier-1 cells resulted in very high ratio (20/20) of positive clones of recombinant adenovirus containing. CD and TK fusion gene as confirmed by PCR test.


CONCLUSIONS
The modified AdEasy system is more convenient and efficient for constructing recombinant adenovirus, which may facilitate further study of anti-tumor therapy targeting at the double suicide gene.
Zheng
Quan
Q
Department of General Surgery, Zhujiang Hospital, First Military Medical University, Guangzhou 510282, China.
Huang
Zong-hai
ZH
Tang
Fu-xiang
FX
Huang
Yuan-yuan
YY
Che
Xiao-yan
XY
D000256:Adenoviridae / Q000235:genetics*; D002122:Calcium Chloride / Q000494:pharmacology; D003587:Cytomegalovirus / Q000235:genetics*; D043525:Cytosine Deaminase / Q000235:genetics*; D015316:Genetic Therapy / Q000379:methods*; D011401:Promoter Regions, Genetic; D011995:Recombination, Genetic; D013937:Thymidine Kinase / Q000235:genetics*; D014170:Transformation, Genetic
D013937:Thymidine Kinase; D043525:Cytosine Deaminase; D002122:Calcium Chloride