Mutagenesis of Viral BACs With Linear PCR Fragments (ET Recombination)
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In the last few years, mutagenesis of viruses with large DNA genomes (like the herpesviruses) was simplified by cloning the viral genomes as bacterial artificial chromosomes (BACs) and their subsequent transfer into
Escherichia coli (E. coli.)
. Owing to the high frequency of restriction sites, classical cloning methods for site-directed mutagenesis are not applicable to these large viral BACs. One possibility for mutagenesis is allele replacement by a two-step recombination procedure (
see
Chapter 18 ). A much more rapid one-step procedure for introduction of mutations into the viral BACs is homologous recombination between a linear DNA fragment and the viral BAC by double crossing-over (
see
principle in Fig. 1 ). Recombination was originally performed with the recombination functions
RecE
and
RecT
and, therefore, termed
ET recombination
or
ET mutagenesis
(
1
). Meanwhile, the recombination functions
red
α
(exo)
and
red
β
(bet)
from bacteriophage λ have been shown to be a good alternative for
RecE
and
RecT
because they are slightly more efficient for double crossing-over. In addition to
red
α/
RecE
and
red
β/
RecT
, expression of the exonuclease inhibitor
red
γ
(gam)
is necessary to allow mutagenesis in bacteria because the gam protein inhibits bacterial exonucleases and protects the linear recombination fragment from degradation. We found that the viral BACs remained more stable when using the
red
recombination functions, in contrast to
RecE
and
RecT
.
Fig. 1.
Principle steps of mutagenesis.
(A)
Preparation of electrocompetent bacterial cells for mutagenesis. Prior to mutagenesis the plasmid pKD46, encoding the recombination functions, is introduced into DH10B containing the viral BAC. In the next step, electrocompetent bacterial cells are prepared by growing the bacteria at 30�C. At the same time, expression of recombinases is induced by addition of 0.1%
l
-arabinose to the growth medium.
(B)
Generation and preparation of the DNA recombination fragment. The DNA recombination fragment that contains a selection marker flanked by homologies to the viral genome (gray boxes) is generated by PCR. The resulting DNA fragment is purified and digested with DpnI to remove residual template DNA.
(C)
Mutagenesis of the viral BAC. For homologous recombination, the DNA recombination fragment is electroporated into the DH10B containing the viral BAC and the expressed recombinases redα, -β, and -γ. After selection with chloramphenicol (Cm) for the BAC and the appropriate antibiotic for the introduced selection marker over night at 37�C, bacteria with the recombinant viral BAC are obtained.
