Posts Tagged ‘Center for Disease Control and Prevention’

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
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 repressilat...

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

There are two types of plasmid integration int...

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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