title: High efficient generation of replication-defective adenoviruses containing thymidine kinase by homogeneous recombination in bacteria.
abstract: 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.
  
  
  CONCLUSIONS
  The replication-defective adenoviruses containing thymidine kinase can be constructed more easily by homogenous recombination in bacteria than conventional techniques.
authors.affiliation: Department of Otolaryngology Head and Neck Surgery, Peking University First Hospital, Beijing, China.
authors.forename: Tie-Chuan
authors.initials: TC
authors.lastname: Cong
authors.forename: Zhe-Ming
authors.initials: ZM
authors.lastname: Lu
authors.forename: Yong
authors.initials: Y
authors.lastname: Li
authors.forename: Li
authors.initials: L
authors.lastname: Zheng
authors.forename: Yong
authors.initials: Y
authors.lastname: Qin
mesh_terms: D000256:Adenoviridae / Q000235:genetics*; D001419:Bacteria / Q000235:genetics*; D003673:Defective Viruses / Q000235:genetics*; D015316:Genetic Therapy / Q000379:methods*; D049452:Green Fluorescent Proteins / Q000235:genetics; D011995:Recombination, Genetic; D013937:Thymidine Kinase / Q000235:genetics*; D014779:Virus Replication
chemical_list: D049452:Green Fluorescent Proteins; D013937:Thymidine Kinase