PAGE 11 OF 12 colonies were correctly targeted and contain the introduced loxP511 site (2 out of 10 clones, a 20% efﬁciency in this experiment). Generation of SW105 and SW106 cells The two DY380-derived bacterial strains EL250 and EL350 contain the defective l prophage needed for recombineering in addition to L-arabinose inducible Flp or Cre genes, respect- ively (11). Both strains have proven to be very useful for BAC modiﬁcation (11), and EL350 is now used routinely for mak- ing conditional targeting vectors for ES cell knock-out experi- ments (4). To further enhance the usefulness of these strains we decided to transfer the galK selection system into them. This was done as previously described for SW102. The galK IS2 element present in both strains was replaced with the wild- type gal promoter and the galK gene in the gal operon deleted using DOG selection. The resulting strains, SW105 (Flp) and SW106 (Cre) (Table 1), were then tested to conﬁrm that they still contained inducible Flp and Cre genes by transforming arabinose-induced and electrocompetent cells with plasmids containing a neo gene ﬂanked by Frt or loxP sites, PL451 and PL452 (4), respectively. Using SW105 or SW106 it is now possible to introduce a point mutation or an informative restriction site into a BAC, retrieve a fragment from this BAC containing the introduced mutation(s) into a plasmid backbone using gap repair, and turn the retrieved fragment into a conditional targeting vector—all using only one bac- terial strain, and only a single initial BAC transformation. DISCUSSION Here, we describe a new recombineering-based E.coli BAC modiﬁcation system that makes use of galK positive selection for growth on galactose minimal medium and galK negative selection (counterselection) for growth on DOG. This modi- ﬁcation system has several advantages compared with the other related BAC modiﬁcation systems. First, the galK selec- tion cassette is small (1231 bp + homology arms) compared with, for example, the sacB–neo cassette (3 kb), making PCR ampliﬁcation and transformation into bacteria easier. Two homology arms are easily added to the galK cassette by includ- ing these sequences in the 50 ends of the primers used for ampliﬁcation (see Materials and Methods). Second, since galK is used for both selection steps, mutations occurring in galK during PCR ampliﬁcation will be selected against during positive selection, signiﬁcantly reducing the risk of DOG resistance from PCR mutations in galK during negative selection. Third, even when very short (33 bp) homology arms were used, the frequency of recombinants among the analyzed DOG-resistant clones was still 20–50%. To our knowledge, successful BAC modiﬁcation with such short homology arms has never been reported. Finally, since galK recombineering is so efﬁcient, little screening is required following selection, reducing the overall hands-on-time to a minimum. galK selec- tion requires growth on minimal medium in both selection steps; although perhaps not used routinely in most molecular biology laboratories where BAC modiﬁcation is needed, these plates are fairly simple to make and should not prevent anyone from using this method. Nucleic Acids Research, 2005, Vol. 33, No. 4 e36 For all such counterselection schemes there will be background since during negative selection any event leading to the loss of the counterselectable marker will result in sur- vival. In our system, virtually all background appears to result from deletions that span the inserted galK gene. These dele- tions likely occur during BAC replication in the 4.5 h out- growth phase, since we have shown that these deletions occur independently of Red induction and in the absence or presence of oligo. It is well established that in recA defective bacteria, BACs are very stable compared with other large insert vectors, such as yeast artiﬁcial chromosomes (YACs). However, using counterselection, rare spontaneous deletions are seen because of the strong selection force. This spontaneous deletion back- ground is not a problem, however, due to the high frequency of homologous recombination obtained with the defective l prophage system. Increasing the length of homology arms used for recombin- eering will reduce the relative number of background deletions observed following negative selection since the percentage of colonies containing BACs that eliminated galK by homolog- ous recombination will be increased. Oligos with longer homology arms can be produced by annealing two oligos with overlapping 30 ends (21). These overlapping oligos (50 single strand overhangs) are then ﬁlled in by DNA polymerase in vivo (or in vitro) and subsequently serve as the substrate for the l Red proteins, exo and bet. Longer homology arms can also be added by traditional cloning using restriction enzymes and DNA ligase (4). A number of uses can be imagined for the strains described here. In principle, our strains can be used to make virtually any kind of BAC modiﬁcation one can imagine, including point mutations or clean deletions or insertions of everything from cDNA to loxP sites or small epitope tags. BAC trimming, the speciﬁc deletion of BAC DNA ﬂanking a gene of interest could also be used to remove genes from BACs prior to making BAC transgenic mice so that only the gene of interest on the BAC is analyzed. This should be of particular interest for BAC rescue experiments in positional cloning. Of course, trimming and modiﬁcation can be combined, since the galK selection cassette can be recycled so that the same BAC can be modiﬁed several times. BAC modiﬁcation could also be the starting point for constructing a gene-targeting vector to allow for more soph- isticated gene targeting in mouse ES cells. The desired muta- tion(s) could ﬁrst be created in the BAC followed by retrieval into a plasmid vector containing a negative-selection marker like thymidine kinase using gap repair (4). Finally, a neo mar- ker could be introduced to allow for positive selection in ES cells. Alternatively, the retrieved fragment could be modiﬁed so that the end result is a conditional targeting vector. Strain SW106, which in addition to galK selection, can be induced to express Cre recombinase, makes it possible to perform all of the steps needed to construct a conditional targeting vector starting with only a single BAC transformation step. Furthermore, in experiments where BAC transgenic mice are used to analyze the effect of deleting long-range regulators of gene expression, galK DOG counterselection can even be used, in the absence of an oligo, to generate a series of dele- tions around the galK insertion site, and the effect on gene regulation studied. The Flp or Cre ORFs contained in strains SW105 and SW106, respectively, can also be replaced for any desired