Seamless assembly of recombinant adenoviral genomes from high-copy plasmids

Jessica J. Miciak, Jason Hirshberg, Fred Bunz

Research output: Contribution to journalArticle

Abstract

The adenoviruses are essential tools for basic research and therapeutic development. Robust methods for the generation of mutant and recombinant viruses are crucial for these diverse applications. Here we describe a simple plasmid-based method that permits highly efficient modification of the adenoviral genome and rapid production of high-titer virus stocks. The 36-kilobase genome of adenovirus serotype 5 was divided into seven tractable blocks that could be individually modified in a single step and reassembled in vitro. Because the system is composed of compact modules, modifications at different loci can be readily recombined. Viral assemblies were delivered to packaging cells by electroporation, a strategy that consistently resulted in the de novo production of 108 infectious units in 3–5 days. In principle, a similar strategy could be applied to any other adenovirus serotype or to other double-strand DNA viruses.

Original languageEnglish (US)
Article numbere0199563
JournalPLoS One
Volume13
Issue number6
DOIs
StatePublished - Jun 1 2018

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Adenoviridae
Viruses
plasmids
Plasmids
Genes
Genome
genome
serotypes
Virus Assembly
DNA viruses
DNA Viruses
Electroporation
electroporation
Product Packaging
viral load
Viral Load
packaging
Packaging
viruses
mutants

ASJC Scopus subject areas

  • Biochemistry, Genetics and Molecular Biology(all)
  • Agricultural and Biological Sciences(all)

Cite this

Seamless assembly of recombinant adenoviral genomes from high-copy plasmids. / Miciak, Jessica J.; Hirshberg, Jason; Bunz, Fred.

In: PLoS One, Vol. 13, No. 6, e0199563, 01.06.2018.

Research output: Contribution to journalArticle

Miciak, Jessica J. ; Hirshberg, Jason ; Bunz, Fred. / Seamless assembly of recombinant adenoviral genomes from high-copy plasmids. In: PLoS One. 2018 ; Vol. 13, No. 6.
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