Abstract
Recombinant adenoviruses provide a versatile system for gene expression studies and therapeutic applications. We have developed an approach that simplifies the generation and production of such viruses called the AdEasy system. A recombinant adenoviral plasmid is generated with a minimum of enzymatic manipulations, employing homologous recombination in bacteria rather than in eukaryotic cells. After transfection of such plasmids into a mammalian packaging cell line, viral production is conveniently followed with the aid of GFP encoded by a gene incorporated into the viral backbone. This system has expedited the process of generating and testing recombinant adenoviruses for a variety of purposes. In this protocol, we describe the practical aspects of using the AdEasy system for generating recombinant adenoviruses. The full protocol usually takes 4–5 weeks to complete.
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Acknowledgements
We apologize to those authors whose original studies were not cited due to space constraints. R.C.H., H.H.L. and T.C.H. were supported by research grants from the American Cancer Society, the Brinson Foundation, and National Institutes of Health. T.C.H. was a recipient of the Outstanding Overseas Young Investigator Collaboration Award from the Natural Science Foundation of China (NSFC no. 30228026) and a recipient of the Bayu Scholar of Chongqing Municipality, Chongqing, China. K.W.K. and B.V. were supported by research grants from the National Institutes of Health and by the Virginia and D.K. Ludwig Fund for Cancer Research. For detailed information, please visit the AdEasy website: http://www.coloncancer.org/adeasy.htm.
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Luo, J., Deng, ZL., Luo, X. et al. A protocol for rapid generation of recombinant adenoviruses using the AdEasy system. Nat Protoc 2, 1236–1247 (2007). https://doi.org/10.1038/nprot.2007.135
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DOI: https://doi.org/10.1038/nprot.2007.135
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