Background Suicide gene therapy is a widely used molecular treatment for head and neck cancer.In this study,we tryto use the method of homogenous recombination in bacteria to clone thymidine kinase gene (tk)-a kind of suicide gene toadenovirus backbone vectors for the construction of replication-defective adenoviruses.Methods pAdTrack-CMV/tk was constructed through subclone of a restriction endonuclease fragment includingthymidine kinase gene from plasmid pCMV-tk to another plasmid pAdTrack-CMV,and then co-transfected withsupercoiled pAdEasy-1,which was an adenoviral backbone vector except for deletions of E1 and E3,to competent E.coil BJ5183 for homogenous recombination using electroporation procedure.With the same method,pAdTrack-CMV wasalso co-transformed with pAdEasy-1 for homogenous recombination in BJ5183、 Identified with restriction endonucleasePacl and polymerase chain reaction(PCR),plasmids pAd-GFP/tk and pAd-GFP were successfully constructed.Each ofthem was digested with Pact and sequently transfected into human embryo kidney 293 cells(HEK293)usingLipofectamine 2000.Results Cornet-like adenovirus-producing loci of Ad-GFP/tk and Ad-GFP were observed after 5 to 7 days of cell cultureAfter twelve days of packaging,the replication-defective adenoviruses were collected.Identified with PCR,thymidinekinase gene was successfully constructed into Ad-GFP/tk.Conclusion The replication-defective adenoviruses containing thymidine kinase can be constructed more easily byhomogenous recombination in bacteria than conventional techniques. 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 toadenovirus backbone vectors for the construction of replication-defective adenoviruses. Methods pAdTrack-CMV / tk was constructed by 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 El and E3, to competent E. coli BJ5183 for homogenous recombination using electroporation procedure. The same method, pAdTrack-CMV wasalso co-transformed with pAdEasy-1 for homogenous recombination in BJ5183, Identified with restriction endonucleasePacl and polymerase chain reaction (PCR), plasmids pAd-GFP / tk and pAd-GFP were successfully constructed. ct and sequently transfected into human embryo kidney 293 cells (HEK293) using Lipofectamine 2000. Results Cornet-like adenovirus-producing loci 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 byhomogenous recombination in bacteria than conventional techniques.