PAGE 7 OF 9 Nucleic Acids Research, 2005, Vol. 33, No. 13 e118 Figure 5. A schematic diagram showing sequencing primers usable after first and second round deletions. Pink rectangles represent the R-end of Tn10 remaining in deletions generated with wild-type transposons such as pTnMarkerless2 (28), and pTn(RSVneo 2)/loxP. Green rectangles indicate the L-end of Tn10 remaining in deletion clones made with any of the loxP511 transposons described here. usually kept intact by propagating them in host strains that are rendered highly recombination deﬁcient (6,7,36). Occasionally, such intra-insert DNA deletions and/or rearrangements can become excessively more frequent than the desirable targeted deletion (P. K. Chatterjee, unpublished data). A second, more important, advantage offered by the loxP transposon-end-deletion procedure is that an entire array of deletions from a particular end of the BAC insert is obtained in a single experiment. This library of truncated BAC-GFP dele- tions can be made without the need to construct new targeting vectors each time. The illustrations in Figures 1 and 3 indicate that sequences ahead of a loxP site are retained in the deleted clone. This characteristic can be used to bring regulatory sequences that are far away, or from another gene, to the near vicinity of the gene of interest during truncations. Exogenous sequences, inserted at suitable sites in the loxP transposons, can be easily engineered into a BAC clone to regulate a gene in novel ways. Studies requiring genes with well deﬁned functional bound- aries are likely to beneﬁt from the procedures described here. They allow isolating a gene free of interfering regulatory elements of an adjacent gene existing in the same BAC clone. The end-deletion technology can be adapted to handle mul- tiple BACs simultaneously. However, results indicate the yield of deletions can be skewed unless BACs with similar growth characteristics are used when pooling. Recombination of loxP sites with Cre protein has been widely used in both bacterial and mammalian systems (19,23,24,26,37–41). The potential for using wild-type and mutant loxP sites in conjunction is even greater. However, the results from a number of recent studies suggest the exclus- ivity between the wild-type and mutant loxP511 sites might be compromised by the level and persistence of Cre protein (19,23–26). Results presented here and those from an earlier study (27) indicate that cross-recombination between wild- type and mutant loxP sites can be reduced if the Cre protein is transiently expressed as in a phage P1 infection. The dif- ferences in speciﬁcity observed between results presented here and those reported by other investigators earlier might be related to the different ways by which loxP sites are exposed to Cre protein in vivo. Although removal of host cell factors and mutations in transposes relax target site speciﬁcity during Tn10 and yeast Ty1 insertions (42), there is yet no evidence to suggest that additional phage encoded and/or induced factors during a P1 infection alters ﬁdelity of Cre recombinase. Thus in the absence of evidence for enhanced speciﬁcity conferred by phage P1 encoded proteins other than Cre, in either bac- terial or mammalian systems (43,44), these results and previ- ous work by others (24,26) appear to support the idea that high levels and/or persistent exposure to Cre protein might in fact contribute to loxP site promiscuity. ACKNOWLEDGEMENTS This work was supported in part by a MBRS-SCORE grant # SO 608049 from the NIGMS, grant # 1U56 CA92077-01 from the NCI, and EXPORT grant #1P20 MD00175-01 from the NIH. We thank Cheryl Harrington, Kathy Wulff, Staris Best, Rosalind Grays, and Dr Eleanor Nunn for support and encour- agement. Funding to pay the Open Access publication charges for this article was provided by National Institute of Health. Conflict of interest statement. None declared. REFERENCES 1. The Mouse Genome Sequencing Consortium. (2002) Initial sequencing and comparative analysis of the mouse genome. Nature, 420, 520–562.