|High efficient generation of replication-defective adenoviruses containing thymidine kinase by homogeneous recombination in bacteria|
|Cong Tie-chuan; Lu Zhe-ming; Li Yong; Zheng Li; Qin Yong|
|关键词||gene therapy adenovirus thymidine kinase|
|刊名||CHINESE MEDICAL JOURNAL|
|WOS标题词||Science & Technology|
|类目[WOS]||Medicine, General & Internal|
|研究领域[WOS]||General & Internal Medicine|
|关键词[WOS]||GENE-TRANSFER ; IN-VIVO ; SYSTEM ; CARCINOMA ; CANCER ; FIBER ; VECTORS ; THERAPY ; CELLS ; HEAD|
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 El 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 Pad and polymerase chain reaction (PCR), plasmids pAd-GFP/tk and pAd-GFP were successfully constructed. Each of them was digested with Pad 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.
|作者单位||1.Peking Univ, Hosp 1, Dept Otolaryngol Head & Neck Surg, Beijing 100034, Peoples R China|
2.Peking Univ, Beijing Inst Canc Res, Dept Genet, Beijing 100036, Peoples R China
|Cong Tie-chuan,Lu Zhe-ming,Li Yong,et al. High efficient generation of replication-defective adenoviruses containing thymidine kinase by homogeneous recombination in bacteria[J]. CHINESE MEDICAL JOURNAL,2007,120(18):1622-1625.|
|APA||Cong Tie-chuan,Lu Zhe-ming,Li Yong,Zheng Li,&Qin Yong.(2007).High efficient generation of replication-defective adenoviruses containing thymidine kinase by homogeneous recombination in bacteria.CHINESE MEDICAL JOURNAL,120(18),1622-1625.|
|MLA||Cong Tie-chuan,et al."High efficient generation of replication-defective adenoviruses containing thymidine kinase by homogeneous recombination in bacteria".CHINESE MEDICAL JOURNAL 120.18(2007):1622-1625.|