[Construction of an antisense RNA recombinant adenovirus vector of the human PDE5A1 gene promoter].
OBJECTIVE
To construct an antisense RNA recombinant adenovirus vector of the human PDE5A1 promoter gene.


METHODS
A cDNA fragment containing the human PDE5A1 promoter and the human PDE5A1-specific exon was determined according to the gene bank. The antisense RNA fragment was synthesized artificially and subcloned into the pENTR. Then, the sequence of pENTR fragment was detected, and the recombinant adenovirus vector pAd/CMV/V5/antisense-PDE5A1 was constructed by LR reaction with the Gateway expression system. The identified recombinant adenovirus plasmid was digested with Pac I and transformed into 293A cells to package recombinant adenovirus particles. The recombinant adenovirus particles were tested with PCR technique and purified after acquisition by CsCl density gradient ultracentrifugation.


RESULTS
The sequencing result showed a 145 bp fragment in pENTR, which was proved to be the domain of the antisense RNA fragment. PCR confirmed that the antisense RNA fragment was cloned into the recombinant adenovirus vector pAd/CMV/V5/antisense-PDE5A1 successfully and could infect 293A cells. The titer of virus stocks was up to 10(8) - 10(10)/microl after purification.


CONCLUSIONS
With the Gateway expression system, the culturing and reproduction of 293A cells can reproduce recombinant adenovirus pAd/CMV/V5-DEST successfully, and the recombinant adenovirus vector can meet the need of in vivo gene transfection in laboratory studies.
Zhao
Yong-Ping
YP
Department of Urology, Peoples Hospital of Peking University, Beijing 100044, China.
Bai
Wen-Jun
WJ
Yan
Zheng
Z
Chen
Guo-Qiang
GQ
Liu
Shi-Jun
SJ
Deng
Qing-Ping
QP
Wang
Xiao-Feng
XF
D015106:3',5'-Cyclic-GMP Phosphodiesterases / Q000235:genetics*; D000256:Adenoviridae / Q000235:genetics*; D002460:Cell Line; D003001:Cloning, Molecular; D054706:Cyclic Nucleotide Phosphodiesterases, Type 5; D005822:Genetic Vectors; D006801:Humans; D010957:Plasmids / Q000235:genetics; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic / Q000235:genetics; D016372:RNA, Antisense / Q000235:genetics*; D011995:Recombination, Genetic
D016372:RNA, Antisense; D015106:3',5'-Cyclic-GMP Phosphodiesterases; D054706:Cyclic Nucleotide Phosphodiesterases, Type 5; C517381:PDE5A protein, human