High efficient generation of replication-defective adenoviruses containing thymidine kinase by homogeneous recombination in bacteria / 中华医学杂志(英文版)
Chinese Medical Journal
; (24): 1622-1625, 2007.
Article
em En
| WPRIM
| ID: wpr-280375
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WPRO
ABSTRACT
<p><b>BACKGROUND</b>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.</p><p><b>METHODS</b>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.</p><p><b>RESULTS</b>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.</p><p><b>CONCLUSION</b>The replication-defective adenoviruses containing thymidine kinase can be constructed more easily by homogenous recombination in bacteria than conventional techniques.</p>
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Base de dados:
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Assunto principal:
Recombinação Genética
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Timidina Quinase
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Bactérias
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Replicação Viral
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Terapia Genética
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Adenoviridae
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Vírus Defeituosos
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Proteínas de Fluorescência Verde
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Genética
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Métodos
Idioma:
En
Revista:
Chinese Medical Journal
Ano de publicação:
2007
Tipo de documento:
Article