Biology Reference
In-Depth Information
recombination, or transposon-based methods. Here, the techniques have often been
pioneered by research labs outside the field of virology and are discussed as they
have been adapted to CMV genetics.
Allelic Exchange by Shuttle Plasmid Mutagenesis
Allelic exchange by shuttle plasmids shares aspects of conventional reverse herpes-
virus genetics. The desired mutation is cloned into a temperature-sensitive suicide
plasmid and flanked by viral sequences homologous to the genomic target site. The
shuttle plasmid is then transformed into the BAC carrying
E. coli
, which expresses
RecA. The exchange between the mutated and
wt
sequence requires two homologous
recombination events (Fig. 2). In the first step, the shuttle plasmid and the BAC
recombine via one homology arm resulting in a co-integrate. At the nonpermissive
Fig. 2
Allelic exchange using shuttle plasmids. A temperature-sensitive suicide shuttle plasmid
that contains the desired mutation (
M
) and viral homologies up- and downstream to the viral target
sequence (
dashed lines
) is transformed into
E. coli
carrying the viral BAC. RecA-mediated
homologous recombination via one homology arm (
A
) leads to cointegrate formation, which is
selected by the antibiotic-resistance genes of the viral BAC and the shuttle plasmid. Free shuttle
plasmids are lost at a nonpermissive temperature. In the second recombination step, the cointegrate
is resolved. Recombination via the same homology arm (A) leads to a wild type (wt) viral BAC
(
on the left
), recombination via the other homology arm (
B
) leads to generation of mutant (M) viral
BAC (
on the right
) Unresolved cointegrates and shuttle plasmids are eliminated by sucrose counter
selection against SacB (
shaded sphere
), which is present in the shuttle plasmid backbone
