Biology Reference
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temperature the nonintegrated shuttle plasmid is lost. Then the selected co-integrate
is resolved by a second recombination event (Fig. 2). There are two possibilities: if
the second recombination occurs via the homology arm of first step,
wt
BAC is
reconstituted. If the second recombination occurs via the other homology arm, a
mutant is gained. Only a minority of the co-integrates undergo RecA-mediated
resolution (O'Connor et al. 1989; Messerle et al. 1997). The inclusion of sacB
(Steinmetz et al. 1983; Blomfield et al. 1991) in the shuttle plasmid allows counter-
selection against unresolved co-integrates. Several herpesvirus mutants have been
generated using this method (Angulo et al. 1998, 2000a, 2000b; Wagner et al. 1999,
2000; Hobom et al. 2000; Brune et al. 2001a; Sanchez et al. 2002). The homologous
recombination mediated by RecA prefers long (1-3 kbp) homologies, which need to
be cloned along with the mutation into the shuttle plasmid. Therefore, the construc-
tion of the shuttle plasmid may need several cloning steps. Shuttle plasmid-based
allelic exchange allows the neat introduction of any kind of mutation (point
mutation, deletion, insertion, sequence replacement) into a viral BAC without
leaving any operational trace in the genome and represents a method of choice when
a complex work is concentrated on one specific genomic region.
Allelic Exchange Using Linear Fragment Mutagenesis
Stewart and colleagues described a one-step mutagenesis method called ET recom-
bination, which uses the recombination functions recET from prophage Rac or the
functions redαβ from bacteriophage λ for introduction of mutations into a circular
DNA by in vitro-generated linear fragments (Zhang et al. 1998; Muyrers et al.
2000). We and others adapted this method to mutagenesis of viral BACs (Adler
et al. 2000; Borst et al. 2001; Kavanagh et al. 2001; Schumacher et al. 2001;
Dorange et al. 2002; Rudolph and Osterrieder 2002; Strive et al. 2002; Tischer
et al. 2002; Wagner et al. 2002). A linear DNA fragment containing a selectable
marker and homologous sequences flanking the target site are transferred into
recombination proficient
E. coli
carrying the target BAC. It is important to prevent
the degradation of the transformed linear DNA. Therefore, either exonuclease-
negative bacteria are used or the exonuclease inhibitor red γ from bacteriophage λ
is co-expressed with the recombinases. The selectable marker along with the muta-
tion is introduced into the BAC by a double crossover event (Fig. 3). Compared to
the RecA-mediated two-step recombination with shuttle plasmids, ET recombina-
tion has advantages. The RecET or redαβ expression allows exact recombination
between homologies as short as 25-50 nts. Therefore, the homology arms including
the mutated sequence can be provided by synthetic oligonucleotide primers, which
are used to amplify the selection cassette. This form of BAC engineering is termed
ET cloning, ET recombination, recombinogenic engineering, or recombineering.
Many systems have been published that use different or altered recombinases
and/or different expression systems controlling their expression. Recombineering
facilitates many kinds of genomic experiments that have otherwise been difficult
