Mutagenesis of Viral BACs With Linear PCR Fragments (ET Recombination)

Abstract

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 redα (exo) and redβ (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 redα/RecE and redβ/RecT, expression of the exonuclease inhibitor redγ (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.