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Fig. 3 Allelic exchange using linear PCR fragments.
a A selectable marker gene ( open box ) that can be
flanked by FRT sites ( black triangle in a square ) is
amplified by PCR using a contiguous primer pair
( arrows ) The primers contain homologies of 35-50 nt to
the viral target sequence at their 5′-ends ( hatched lines ),
a mutation ( M ) and priming regions to the selectable
marker gene ( black lines ) b The generated linear PCR
fragment is transformed into E. coli carrying the viral
BAC and containing recombinases and an exonuclease
inhibitor (redαβγ). The desired mutation along with the
selectable marker and the FRT sites is introduced into the
viral BAC by double crossover. c Additional expression
of the site-specific recombinase FLP leads to the exci-
sion of the selectable marker reducing the operational
sequences to only one FRT site
to carry out. The mutagenesis is independent of specific sequence elements; thus
the site of mutagenesis can be freely chosen. A risk is the instability of the viral
BAC during this mutagenesis procedure since presence of even short repeated
sequences in the target genome can lead to unwanted recombination events.
Replacing wt sequences with a positive selection marker requires only one recom-
bination step and it is easy to create knockout mutants. In addition, it operates
practically without background because the positive selection allows the survival
of only the desired recombinants and directly sorts out the genome rearrangements
induced by the repeat regions. Recently, a comprehensive set of individual deletion
mutants of all HCMV genes have been generated by recombineering for functional
profiling of the entire genome (Yu et al. 2003). However, this procedure leaves
operational trace in the mutated genomes, namely the bacterial selection marker,
which is associated with the risk of unpredictable polar effect on usually complex
viral transcription units. To lower the size and the risk of the mutational traces, the
selectable marker can be flanked with FRT (FLP recognition target) sites
(Cherepanov and Wackernagel 1995). These sites allow excision of the marker
in a second step by Flp recombinase in E. coli (Fig. 3c), leaving only approxi-
mately 70-100 nt extra sequence around the introduced mutation (Wagner and
Koszinowski 2004).
From the very beginning of BAC recombineering, attempts were made to con-
struct mutants without operational trace. All strategies that are useful for the size
of herpesvirus BACs apply two consecutive steps of homologous recombination.
First, a combined marker, which allows both positive and counter-selection in
E. coli , is introduced at the targeted site, resulting in an intermediate that is
isolated by positive selection. Next, by a second round of recombination, the
markers are replaced with the desired sequence and the right recombinant can be
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