e118 Nucleic Acids Research, 2005, Vol. 33, No. 13 identical in size to starting BACs (28). DNA from 20 clones of each deleted series was sequenced directly using a transposon- end primer (20) and Big dye terminator chemistry on an ABI- 3100 AVANT genetic analyzer. Primer extended products were puriﬁed using Magnesil (Promega Corporation) accord- ing to procedures supplied by the manufacturer and described in (27,28). Primers for sequencing newly created ends of dele- tions from the loxP511 side of insert DNA, after trimming the wild-type end, have been developed. These are listed below: Sequencing primers Seq 8: d (GCAGTGTGACCGTGTGCTTCTCAAATGC); Seq 21: d (GATCGGCGCGCCATGATC); Seq 25: d (GCCAGTAGTCGTGACTG); Neo 8: d (GTTTTCCCTTG- TCCAGATAGC); Neo 11: d (CTGAGTGCTTGCGGCA- GCG); Neo 12: d (GAATCGTTTTCCGGGACG); Neo 16: d (GATCTCATGCTGGAGTTCTTCGCC). RESULTS Truncating DNA inserts from both ends using loxP transpo- sons critically depends on the degree of leakage in recomb- ination between the two loxP sites ﬂanking the insert. Because varying degrees of recombination cross-reactivity between these sites was observed in previous studies (19,23–26), it was important to reinvestigate this under the P1 transduction conditions that are actually used in our deletion procedure (27). Estimating recombination cross-reactivity between wild-type and mutant loxP511 sites using phage P1 transduction Several mutant loxP sites have been tested both for their recombination efﬁciency and exclusivity under different Cre protein-exposure conditions (23–27). Phage P1 expresses Cre early during an infection and efﬁciently transduces a wild- type loxP plasmid with high ﬁdelity (30–32). The ability of the wild-type loxP site in phage P1 to transduce the mutant loxP511 plasmid was used as an indicator of recombination cross-reactivity between these sites (27). Results in Table 1 indicated that although P1 was able to efﬁciently transduce the plasmid with a wild-type site, it could recover the mutant loxP511 plasmid only very inefﬁciently (compare #17 with #21). Unlike the results with two other mutant loxP sites described earlier (27), inhibition of transduction of the Table 1. P1 transduction of plasmids carrying wild type and/or loxP511 sites. Clone #17 #21 #39 #45 Transformed clones #39/21 #45/21 Wt loxP loxP511 a+++++, 1000 colonies/plate. b+, 5 colonies/plate. Size (kb) 12 8 40 60 ATAACTTCGTATA GCATACAT TATACGAAGTTAT ATAACTTCGTATA GTATACAT TATACGAAGTTAT PAGE 4 OF 9 loxP511 mutant was incomplete. Comparing parallel transduc- tions we ﬁnd that the loxP511 plasmid is transduced 0.1–0.5% as efﬁciently as the wild-type. This level of recombination cross-reactivity is signiﬁcantly less than that which was repor- ted earlier in several in vivo and in vitro analyses (19,23–26). The exclusivity between the two sites appears high, although not absolute as reported originally (33). Consistent with pre- vious ﬁndings (27), phage P1 was able to efﬁciently transduce the loxP511 plasmid if another carrying both a wild-type and the loxP511 site exists in the cell (rows 5, 6, Table 1). Phage P1 transduced the loxP511 plasmid in the presence of BAC deletions, #39 and #45, generated from BAC RP23-444K15 by truncating from the wild-type loxP end with a markerless wild-type loxP transposon (28). Generating deletions from the loxP511 end of insert DNA in pBACe3.6 Because the 511 and wild-type loxP sequences cross- recombine only slightly, insert DNA ﬂanked by these can be deleted from both ends using transposons carrying those sites. Verifying authenticity of the newly created ends after each round of deletion should compensate for the minor leaki- ness. Transposons were constructed with the two types of loxP sites such that a different end of the Tn10 was left behind after deletion formation at each end of insert DNA. Unique primers were designed into each transposon-end remaining after the recombination. Two classes of transposons were constructed carrying the loxP511 site: (i) markerless and (ii) with kanamycin resistance gene as marker. Only transposons of the ‘B’ series of both classes, with the loxP511 oriented as shown in Figure 1, were used in this study. The ‘A’ series of both classes had the loxP511 site in the opposite orientation. As noted earlier (28), transposition of a markerless loxP transposon is selected through P1-headful packaging and not by the transfer of an antibiotic resistance gene into the target DNA. This necessit- ates the starting BAC to be larger than P1-headful size. Depending on which end of the genomic insert is deleted ﬁrst, the markerless transposon could carry a wild-type or a mutant loxP511 site. Markerless transposons with wild-type loxP sites have been previously described (28). The transposon plasmid pTnLoxP511(B)markerless 1 was transformed into each of the 10 BACs listed in Materials and Methods. Transformed colonies were selected on LB agar plates containing chloramphenicol plus ampicillin. Colonies Marker camR kanR camR camR LoxP site Wild-type loxP only loxP511 only Wild-type plus loxP511 Wild-type plus loxP511 After transduction with phage P1 kan cam +b +++++a +++++a +++++a +++++a +++++a +++++a +++++a