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Cloning the Vaccinia Virus Genome as a Bacterial Artificial Chromosome

Curator: Larry H Bernstein, MD, FCAP

Cloning the vaccinia virus genome as a bacterial artificial chromosome in Escherichia coli and recovery of infectious virus in mammalian cells

A Domi and B Moss

PNAS  Sep 17, 2002; 99(19):12415–12420     http://www.pnas.org/dx.cgi.doi/10.1073/pnas.192420599

http://www.pnas.org/Cloning_the_vaccinia_virus_genome_as_a_bacterial_artificial_chromosome_in_Escherichia_coli_and_recovery_of_infectious_virus_in_mammalian_cells/

The ability to manipulate the vaccinia virus (VAC) genome,

• as a plasmid in bacteria,
• would greatly facilitate genetic studies and
• provide a powerful alternative method of making recombinant viruses.

VAC, like other poxviruses, has a linear, double-stranded DNA genome with covalently closed hairpin ends that are resolved

• from transient head-to-head and tail-to-tail concatemers
• during replication in the cytoplasm of infected cell.

Our strategy to construct a nearly 200,000-bp VAC-bacterial artificial chromosome (BAC) was based on

• circularization of head-to-tail concatemers of VAC DNA.

Cells were infected with a recombinant VAC containing inserted sequences for plasmid replication and maintenance in Escherichia coli; DNA concatemer resolution was inhibited

• leading to formation and accumulation of head-to-tail concatemers,

in addition to the usual head-to-head and tail-to-tail forms;

• the concatemers were circularized
  • by homologous or Cre–loxP-mediated recombination; and
• E. coli were transformed with DNA from the infected cell lysates.

Stable plasmids containing the entire VAC genome, with an intact concatemer junction sequence, were identified. Rescue of infectious VAC was consistently achieved

• by transfecting the VAC–BAC plasmids into mammalian cells that were infected with a helper nonreplicating fowlpox virus.

The plasmids used to implement the repressilator in Escherichia coli. (Photo credit: Wikipedia)

There are two types of plasmid integration into a host bacteria: Non-integrating plasmids replicate as with the top instance, whereas episomes, the lower example, integrate into the host chromosome. (Photo credit: Wikipedia)

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Posted in Genome Biology, Infectious Disease & New Antibiotic Targets, International Global Work in Pharmaceutical, Molecular Genetics & Pharmaceutical, Pharmaceutical Industry Competitive Intelligence, Technology Transfer: Biotech and Pharmaceutical | Tagged Center for Disease Control and Prevention, DNA, Escherichia coli, Morbidity and Mortality Weekly Report, Proceedings of the National Academy of Sciences of the United States of America, San Diego, Smallpox, Vaccinia | 3 Comments

3 Responses

1. on March 4, 2013 at 10:48 PM | Reply Transphosphorylation of E-coli proteins and kinase specificity | Pharmaceutical Intelligence

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2. on March 5, 2013 at 6:22 AM | Reply RNA virus genome as bacterial chromosome | Pharmaceutical Intelligence

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3. on March 5, 2013 at 7:07 AM | Reply Malaria genomes | Pharmaceutical Intelligence

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