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Designing Plasmid Vectors

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Gene Therapy of Cancer

Part of the book series: Methods in Molecular Biology™ ((MIMB,volume 542))

Summary

Nonviral gene therapy vectors are commonly based on recombinant bacterial plasmids or their derivatives. The plasmids are propagated in bacteria, so, in addition to their therapeutic cargo, they necessarily contain a bacterial replication origin and a selection marker, usually a gene conferring antibiotic resistance. Structural and maintenance plasmid stability in bacteria is required for the plasmid DNA production and can be achieved by carefully choosing a combination of the therapeutic DNA sequences, replication origin, selection marker, and bacterial strain. The use of appropriate promoters, other regulatory elements, and mammalian maintenance devices ensures that the therapeutic gene or genes are adequately expressed in target human cells. Optimal immune response to the plasmid vectors can be modulated via inclusion or exclusion of DNA sequences containing immunostimulatory CpG sequence motifs.

DNA fragments facilitating construction of plasmid vectors should also be considered for inclusion in the design of plasmid vectors. Techniques relying on site-specific or homologous recombination are preferred for construction of large plasmids (>15 kb), while digestion of DNA by restriction enzymes with subsequent ligation of the resulting DNA fragments continues to be the mainstream approach for generation of small- and medium-size plasmids. Rapid selection of a desired recombinant plasmid against a background of other plasmids continues to be a challenge. In this chapter, the emphasis is placed on efficient and flexible versions of DNA cloning protocols using selection of recombinant plasmids by restriction endonucleases directly in the ligation mixture.

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Authors and Affiliations

  1. Molecular Medicine, National Heart and Lung Institute, Faculty of Medicine, Imperial College London, Sir Alexander Fleming Building, Exhibition Road, London, SW7 2AZ, UK

    Oleg Tolmachov

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  1. Oleg Tolmachov
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Correspondence to Oleg Tolmachov .

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Editors and Affiliations

  1. Molecular Medicine (MDC), Max Delbrück Center for, Robert-Rössle-Str. 10, Berlin, 13125, Germany

    Wolfgang Walther

  2. Molecular Medicine (MDC), Max Delbrück Center for, Robert-Rössle-Str. 10, Berlin, 13125, Germany

    Ulrike S. Stein

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© 2009 Humana Press, a part of Springer Science+Business Media, LLC

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Tolmachov, O. (2009). Designing Plasmid Vectors. In: Walther, W., Stein, U. (eds) Gene Therapy of Cancer. Methods in Molecular Biology™, vol 542. Humana Press. https://doi.org/10.1007/978-1-59745-561-9_6

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  • DOI: https://doi.org/10.1007/978-1-59745-561-9_6

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  • Publisher Name: Humana Press

  • Print ISBN: 978-1-934115-85-5

  • Online ISBN: 978-1-59745-561-9

  • eBook Packages: Springer Protocols

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