Results

To make subtle changes in the nucleotide sequence of a BAC, such as the introduction of a point mutation, we developed a two‐step use of ET recombination. Both steps were performed using the SacB–neo fusion protein present on pSacB‐neo (Figure 1A). In the first step, selection pressure was applied for expression of the Neo part of the fusion protein, and in the second step, selection pressure was applied against the SacB part. Figure 1B shows an example of the strategy, in which a single G nucleotide was introduced directly upstream of an AATTC sequence present in a BAC of> 100 kb, which carries the mouse Af‐4 gene (Baskaran et al., 1997). As previously described (Zhang et al., 1998; Muyrers et al., 1999), the E. coli host carrying the Af‐4 BAC was capacitated for ET recombination by transforming it with a plasmid carrying the ET recombination genes. Here we used pBAD‐RedGam (Figure 1A), which contains redα/redβ under the l‐arabinose‐inducible promoter and from which gam is constitutively expressed to inhibit endogenous RecBC activity (Murphy, 1991).