e36 Nucleic Acids Research, 2005, Vol. 33, No. 4 PAGE 8 OF 12 Figure 4. BAC trimming using galK selection. (A) Illustration of the design of the deletion experiment. Homology arm 1 (H1) was held constant, and H2 was separated from H1 by either 50, 75 or 100 kb. (B) SpeI restriction analysis of BAC miniprep DNA from 12 clones showing deletions of 50, 75 and 100 kb, respectively, after the insertion of the galK selection cassette. The first lane is unmodified RP23-341F12 BAC DNA, which was included as a control. All tested clones had the intended deletion. removed with an oligo using galK negative selection to cleanly delete the galK cassette from the BAC as described in the previous experiment (data not shown). Insertion of a mutant loxP511 site into a BAC Encouraged by the efﬁciency of this selection system, we decided to see whether we could introduce a single 34 bp loxP511 site cleanly into BAC DNA using galK positive/neg- ative selection. It has been previously shown that loxP and loxP511 sites cannot recombine with each other; therefore, the introduced loxP511 site can only recombine with the loxP511 site present in the BAC vector backbone and not the wild-type site (Figure 5A) (20). We used galK positive selection to insert a PCR-ampliﬁed galK cassette ﬂanked by 50 bp homology arms to a position 95 kb away from the mutant loxP511 site in the BAC RP23-341F12 vector backbone (Figure 5A). We then replaced the galK cassette using DOG counterselection with a double-stranded 100 bp oligo containing a 34 bp loxP511 site ﬂanked by two 33 bp homology arms. In this experiment, we observed less than a 10-fold difference in the number of col- onies on the plates from heat-induced and non-induced bac- teria, suggesting fewer recombinants. This lower frequency of recombinants, when compared with the Nras G12D substitu- tion experiment, is likely explained by the shorter homology arms used in this latter experiment, as it has been shown that the efﬁciency of recombination increase four orders of mag- nitude when homology length is increased from 20 to 40 bp (10). We analyzed six BAC minipreps from potential recom- binants by digesting with SpeI and comparing the restriction patterns with that of wild-type RP23-341F12 BAC DNA (Figure 5B). Three of six (50%) colonies had exactly the same restriction pattern as the unmodiﬁed RP23-341F12 BAC, suggesting that DOG resistance had selected for the desired homologous recombination products in these three cases, whereas the other three clones apparently became DOG resistant due to the selection of BACs that carry large deletions spanning galK. The 100 bp oligo was designed so that the loxP511 site would be inserted in the same orientation as the loxP511 site in the BAC vector backbone. To conﬁrm that the BACs with the wild-type restriction pattern (3, 5 and 6) had the loxP511 site correctly inserted, we electroporated these three BACs into electrocompetent and L-arabinose induced EL350 cells, which carry an L-arabinose-inducible Cre gene, and then plated the cells on LB plates containing chlor- amphenicol to select for the BACs. Two colonies generated from each recombinant loxP511 clone were then tested by SpeI digestion of BAC miniprep DNA, and all had the expec- ted, Cre-mediated, 95 kb deletion (Figure 5C). Recombination was conﬁrmed by PCR using a forward primer from the BAC vector backbone and a reverse primer mapping to a position distal to the reverse homology arm used to insert the galK cassette. These primers are 95 kb apart on wild-type RP23- 341F12 DNA and only 378 bp apart on Cre/loxP511 recom- bined DNA. PCR analysis of all six clones produced a band of the expected length, whereas no product could be ampliﬁed from unmodiﬁed RP23-341F12 BAC DNA (Figure 5D). Source of background deletions following DOG selection In the Nras G12D substitution and loxP511 insertion experi- ments, a background of DOG-resistant bacteria, which lacked the desired mutation and instead carried unwanted deletions that spanned the galK selection cassette, were observed. The relative ratio of colonies containing BACs with deletions was also higher when short homology arms were used. This was expected since homologous recombination is more efﬁcient with longer homology arms (10) that would increase the rel- ative frequency of correctly targeted BACs. The source of the deletions could be l Red-mediated, since proteins capable of mediating recombination are expressed when the Red genes