PMID- 17908483
OWN - NLM
STAT- MEDLINE
DCOM- 20071108
LR  - 20121115
IS  - 0366-6999 (Print)
IS  - 0366-6999 (Linking)
VI  - 120
IP  - 18
DP  - 2007 Sep 20
TI  - High efficient generation of replication-defective adenoviruses containing 
      thymidine kinase by homogeneous recombination in bacteria.
PG  - 1622-5
AB  - BACKGROUND: Suicide gene therapy is a widely used molecular treatment for head 
      and neck cancer. In this study, we try to use the method of homogenous 
      recombination in bacteria to clone thymidine kinase gene (tk)-a kind of suicide 
      gene to adenovirus backbone vectors for the construction of replication-defective 
      adenoviruses. METHODS: pAdTrack-CMV/tk was constructed through subclone of a 
      restriction endonuclease fragment including thymidine kinase gene from plasmid 
      pCMV-tk to another plasmid pAdTrack-CMV, and then co-transfected with supercoiled 
      pAdEasy-1, which was an adenoviral backbone vector except for deletions of E1 and 
      E3, to competent E. coli BJ5183 for homogenous recombination using 
      electroporation procedure. With the same method, pAdTrack-CMV was also 
      co-transformed with pAdEasy-1 for homogenous recombination in BJ5183. Identified 
      with restriction endonuclease PacI and polymerase chain reaction (PCR), plasmids 
      pAd-GFP/tk and pAd-GFP were successfully constructed. Each of them was digested 
      with PacI and sequently transfected into human embryo kidney 293 cells (HEK293) 
      using Lipofectamine 2000. RESULTS: Comet-like adenovirus-producing foci of 
      Ad-GFP/tk and Ad-GFP were observed after 5 to 7 days of cell culture. After 
      twelve days of packaging, the replication-defective adenoviruses were collected. 
      Identified with PCR, thymidine kinase gene was successfully constructed into 
      Ad-GFP/tk. CONCLUSION: The replication-defective adenoviruses containing 
      thymidine kinase can be constructed more easily by homogenous recombination in 
      bacteria than conventional techniques.
FAU - Cong, Tie-Chuan
AU  - Cong TC
AD  - Department of Otolaryngology Head and Neck Surgery, Peking University First 
      Hospital, Beijing, China.
FAU - Lu, Zhe-Ming
AU  - Lu ZM
FAU - Li, Yong
AU  - Li Y
FAU - Zheng, Li
AU  - Zheng L
FAU - Qin, Yong
AU  - Qin Y
LA  - eng
PT  - Journal Article
PT  - Research Support, Non-U.S. Gov't
PL  - China
TA  - Chin Med J (Engl)
JT  - Chinese medical journal
JID - 7513795
RN  - 147336-22-9 (Green Fluorescent Proteins)
RN  - EC 2.7.1.21 (Thymidine Kinase)
SB  - IM
MH  - Adenoviridae/*genetics
MH  - Bacteria/*genetics
MH  - Defective Viruses/*genetics
MH  - Genetic Therapy/*methods
MH  - Green Fluorescent Proteins/genetics
MH  - *Recombination, Genetic
MH  - Thymidine Kinase/*genetics
MH  - Virus Replication
EDAT- 2007/10/03 09:00
MHDA- 2007/11/09 09:00
CRDT- 2007/10/03 09:00
PHST- 2007/10/03 09:00 [pubmed]
PHST- 2007/11/09 09:00 [medline]
PHST- 2007/10/03 09:00 [entrez]
PST - ppublish
SO  - Chin Med J (Engl). 2007 Sep 20;120(18):1622-5.