Abstract
Recombinant adenoviruses are widely used for delivery and expression of transgenes for therapeutic purposes and in fundamental research. AdEasy™ adenoviral vector system allows for rapid and efficient generation of replication-deficient adenoviruses. One important crucial step in producing recombinant adenoviruses is the generation of a recombinant adenoviral plasmid in E. coli BJ5183 cells via homologous recombination between the transgene encoding shuttle vector and the pAdEasy-1 plasmid, which carries a significant part of human adenovirus serotype 5 (Ad5) genome. In many published protocols, the necessity of electroporation for the transformation of the shuttle vector and pAdEasy-1 into E. coli cells is emphasized, thus making necessary the availability of the appropriate instruments and consumables. Here, we have proposed an optimized protocol for the generation of recombinant adenoviruses with the use of chemically competent E. coli cells only, without the use of electroporation. We have demonstrated the efficient transformation of linearized nonpurified shuttle vector into chemically competent E. coli BJ5183-pAdEasy-1 cells allowing identification of clones with correct recombinant adenoviral plasmids. Using the optimized protocol, we have generated four functional replication-deficient adenoviruses expressing target proteins in HEK293 cells. The protocol that has been suggested provides a significantly simplified and cheaper method for the generation of recombinant adenoviruses due to lack of requirement to use instruments and consumables for electroporation.
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Funding
This work was financially supported by the Russian Foundation for Basic Research (projects nos. 16-04-00686 and 16-04-00376) and supported by the infrastructure of the Living Cell Biology and Biomedical Drug Nanotransporters Core Facilities of the Institute of Gene Biology Russian Academy of Sciences.
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The authors declare that they have no conflict of interest. This article does not contain any studies involving animals or human participants performed by any of the authors.
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Translated by P. Vikhreva
Abbreviations: kbp—kilobase pairs, CFU–colony-forming unit.
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Shepelev, M.V., Korobko, I.V. An Optimized Electroporation-Free Method for Generation of Recombinant Adenoviral Plasmids. Mol. Genet. Microbiol. Virol. 34, 228–233 (2019). https://doi.org/10.3103/S0891416819040098
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DOI: https://doi.org/10.3103/S0891416819040098