PAGE 7 OF 12 Nucleic Acids Research, 2005, Vol. 33, No. 4 e36 Figure 3. Introduction of a G12D mutation in the Nras gene. (A) SpeI restriction analysis of BAC miniprep DNA. First lane is the unmodified CITB-50J2 Nras BAC. Lanes 1–12 show digestion patterns of 12 clones counterselected for the substitution of galK with an oligo containing the G!A substitution for the second position of codon 12 of Nras. Clones 7 and 10 had internal deletions, indicating that DOG resistance was achieved by spontaneous deletion and not homologous recombination. These two clones were not analyzed further. (B) Sequence analysis of a PCR product spanning the modified region from clones 1–6, 8–9 and 11–12. All clones had the intended substitution (highlighted). However, clones 9 and 11 also had an internal basepair deletion indicated by a minus (highlighted). The Nras ATG and codon 12 are indicated (shadow). by sequencing of the modiﬁed region of the 10 BAC clones (Figure 3B). All 10 sequenced clones had the desired point mutation. The effective recombination efﬁciency in this experiment, however, was 8/12 (67%) since 2 of the 10 clones also had an additional single base pair deletion (clones 9 and 11). These deletions probably occurred during oligo synthesis, since we did not purify the oligos beyond desalting (19). Because of the high efﬁciency in this system, there was no need to pre-screen the selected colonies prior to picking for minipreps. Large BAC deletions A drawback of using BACs for the production of transgenic mice is the frequent presence of other genes on the BAC in addition to the gene of interest. This is especially a problem for BAC complementation used in positional cloning, where a BAC is tested for its ability to rescue a loss-of-function mutant phenotype by making BAC transgenic mice. If complementa- tion is achieved, it is impossible to know which gene on the BAC is responsible for the rescue. Therefore, we decided to see whether galK selection could be used to make large, speciﬁc and clean deletions in BACs so as to remove unwanted genes (a process called BAC trimming). As a model system, we chose a mouse BAC from the C57BL/6-derived library RP23, RP23-341F12, since we could obtain the BAC sequence directly from the UCSC genomic web browser (see Materials and Methods). We then used PCR to amplify the galK selec- tion cassette with primers containing homology to the BAC. The forward primer contained 50 bp of homology to the very 50 end of the BAC insert, and this primer was combined with three different reverse primers, having 50 bp of homology to positions 50, 75 and 100 kb away from the forward primer’s homology (Figure 4A). SW102 bacteria containing the 341F12 BAC were heat-induced and made electrocompetent, and then transformed with the three different selection cassettes, fol- lowed by the selection on minimal medium with galactose. From the many resulting Gal+ colonies, we picked four from each experiment and then did a SpeI restriction analysis of the BAC miniprep DNAs from them (Figure 4B). All four colon- ies from each experiment had the desired deletion (50, 75 and 100 kb, respectively). The galK selection cassette can then be