e118 Nucleic Acids Research, 2005, Vol. 33, No. 13 Because regulatory elements can sometimes be within introns or located downstream of a gene, it would be desirable to be able to truncate BAC inserts from both ends. Fortunately cloning vehicles, such as the pBACe3.6 and pTARBAC1 or pTARBAC2 vectors, used for the newer resource libraries contain the insert DNA ﬂanked by two different loxP sites (22). An arrangement such as this might have been considered ideal for deleting DNA from both ends with transposons carrying the different loxP sites had results from several subsequent studies not discouraged developing such strat- egies: cross-recombination between wild-type and the mutant loxP511 site that ﬂanked DNA inserts in these clones, have been reported to range from 5 to 100% in a variety of settings (19,23–26). A recent study using a pair of different loxP site mutants suggests the earlier ﬁndings might have been inﬂuenced by excessive and/or sustained levels of Cre protein from constitutive expression, during recombination (27). Cross- recombination between wild-type and loxP511 sites was there- fore, reinvestigated using transient expression of Cre protein which occurs during phage P1 transduction. We now report that the leakage in recombination between these two sites was found to be not more than 0.5% of that between two wild-type sites. The results enabled us to progressively delete both ends of genomic inserts in pBACe3.6 and related vectors using transposons that carry either a wild-type or a mutant loxP511 site. MATERIALS AND METHODS The oligodeoxyribonucleotides, d (GGCCGCTAATAACTT- CGTATAGTATACATTATACGAAGTTATTGTTTAAACC) and d (GGCCGGTTTAAACAATAACTTCGTATAATGTA- TACTATACGAAGTTATTAGC), were annealed by heating equimolar quantities in 10 mM Tris–HCl, pH 8.0, buffer containing 1 mM EDTA and 10 mM NaCl to 70 C and slow cooling to room temperature. The duplex oligo contains the mutant loxP511 sequence along with a PmeI site, and was ligated to plasmid DNA linearized with NotI enzyme. Construction of Tn10 minitransposons with mutant loxP511 sites The transposon plasmid pTnloxP*-1 (27), was the starting point for all mutant loxP511 site-containing plasmids. The gene for chloramphenicol resistance located near one of the 70 bp inverted repeat ends was removed by digesting the pTnloxP*-1 DNA to completion with PvuII, gel purifying the largest fragment, and performing a partial digest with ScaI. The largest fragment was again gel puriﬁed, and the two blunt ends created by the PvuII and ScaI enzymes ligated with high concentration T4 DNA ligase. Transformed colonies were selected on LB agar plates with ampicillin. The gene for ampicillin resistance is located outside the 70 bp inverted repeat ends of the transposon. Next the sequences for tetracycline resistance gene and the mutant loxP*-1 site were removed by NotI digestion, and it was ligated with a duplex oligonucleotide with NotI compat- ible ends that contained the mutant loxP511 sequence and a PmeI restriction site (shown above). Plasmids acquiring the loxP511 oligonucleotide were screened for by digesting clone DNA with PmeI enzyme. Both orientations of the loxP511 site-inserted transposon were isolated. They were named pTnLoxP511(A)markerless 1 and pTnLoxP511(B)- markerless 1. They are designated markerless because they do not contain any antibiotic resistance markers within the 70 bp inverted repeat ends of the transposon to select for transpositions of the loxP511 site into target DNA [see discussion (28)]. An additional oligonucleotide duplex for anchoring a sequencing primer (Seq 25) was inserted into the unique AscI site of pTnLoxP511(B)markerless 1 to generate pTnLoxP511(B)markerless 2. The plasmid was linearized with AscI and the duplex obtained by hybridizing the follow- ing oligonucleotides M13C and M13D was ligated as described above. M13C: d (CGCGCCTAGTAAAACGACGGCCAGTAG- TCGTGACTGGGAAAACCTGTTTAAACC); M13D: d (CGCGGGTTTAAACAGGTTTTCCCAGTCACGACTACT- GGCCGTCGTTTTACTAGG). A RSV neomycin resistance gene cassette, coding for kana- mycin resistance in bacteria, was excised from pTnBAC/loxP (14) with AscI, and inserted into the AscI site of pTnLox- P511(B)markerless 1 (Figure 1). The resulting transposon Wt & loxP511 Transposon plasmids transposase loxP511 Pme I Asc I L ori amp r pTnLoxP511(B)markerless 1 transposase Pme I wt loxP Not I Asc I Bgl II Asc I Not I PAGE 2 OF 9 Not I R R Not I R ori amp r L RSVneo < 1.7 kb > pTn(RSVneo 2)/loxP transposase Asc I Asc I Pme I Not I loxP511 ori amp r L <RS1.V7nkebo > pTnLoxP511(B)RSVneo 2 Figure 1. New transposon plasmids containing wild-type and loxP511 sites. A schematic representation of transposon plasmids used for deleting both ends of insert DNA cloned in the pBACe3.6 and related vectors pTARBAC1 and pTARBAC2. The transposon plasmid pTnLoxP511(B)markerless 1, shown in the top panel, has no antibiotic resistance marker within the transposing part marked by the small rectangular boxes colored pink or green. Bottom panel shows a kanamycin resistance gene-containing variant, pTnLoxP511(B)RSV- neo 2. The middle panel shows a wild-type loxP transposon plasmid with kanamycin resistance gene. Wild-type loxP site is shown with the thick con- tinuous arrow, while the mutant loxP511 sites are indicated by bold broken arrows. The pink end R remains in deletion clones generated with the wild-type loxP transposons, while the green end L is left behind in deletions with the loxP511 transposons.