e118 Nucleic Acids Research, 2005, Vol. 33, No. 13 PAGE 6 OF 9 Deleting From the Wt-loxP End of DNA insert in pBACe3.6 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 80- 70- 60- 50- 40- 30- 20- 10- 5- Figure 4. FIGE analysis of NotI digested DNA isolated from deletions made from the wild-type loxP end. Left panel: DNA isolated from deletion clones generated with pTnMarkerless2 (28) in BAC RP23-92L23 is shown in lanes 1–13. Lanes 16–30 show DNA from first round deletions generated with pTn(RSVneo 2)/loxP in clone RP23-444K15. Lane 15 contains DNA from starting BAC RP23-444K15. Lane 14 shows a 5 kb DNA ladder. The size of BAC vector DNA fragment is 6.8 and 8.4 kb with pTnMarkerless2 and pTn(RSVneo 2)/loxP, respectively. The starting BAC produces a 8.8 kb BAC vector band (lane 15). transposon. Preference as to which end of insert DNA, the wild-type or the loxP511, will be truncated ﬁrst dictates the choice of markerless transposon: pTnMarkerless2 [with wild- type loxP (28)] or pTnLoxP511(B)markerless 1 (Figure 1). A second round of truncations with pTn(RSVneo 2)/loxP was made with the largest clone in the deletion series obtained with pTnLoxP511(B)markerless 1 (shown in lanes 3 and 31 of Figure 2). NotI digested DNA isolated from clones obtained in the second round of deletions is shown in lanes 32–40, Figure 2. Because the starting clone here was <110 kb, both deletions and inversions were isolated in about equal numbers (14,29). Inversions are shown in lanes 42–48 (Figure 2). Similar truncations from both ends were also made by delet- ing ﬁrst from the wild-type loxP end of inserts with pTnMark- erless2, and then using pTnLoxP511(B)RSVneo 2 to trim the other end of a clone isolated in the ﬁrst round deletions (data not shown). Processing a mixture of BAC clones transformed with transposon plasmid to increase throughput of making end-deletions Multiple BACs can be processed together as a mixture in a single tube to make end-deletions. Because clones tend to have different growth characteristics, this is best achieved by transforming each clone separately with the desired trans- poson plasmid and growing them to saturation before pooling. As many as 32 BACs were transformed in parallel. Aliquots from nine such tansformed BAC cultures were pooled, and processed for deletions exactly as described earlier (29). For simplicity of sequencing downstream, BACs in a pool were transformed with the same Tn-plasmid. Each deletion was seq- uenced with primers speciﬁc to both ends of the insert DNA remaining after the deletion i.e. one from the transposon, the other speciﬁc to the opposite unaltered end of insert DNA (SP6 or T7). The latter sequence helped identify the parent from which the deletion was obtained. Analysis of the results indic- ates although each of the nine BACs is represented in deletions, the distribution is quite skewed: deletions from clone RP23- 444K15 dominate 4-fold over those from RP11-219A15, RP23-209O22 or RP23-92L23, although all four were equally efﬁcient in producing deletions when processed individually. Sequencing ends of deletions with primers from the wild-type or loxP511 transposons Transposon-ends marked R (pink) and L (green) shown in Figures 1, 3 and 5 remain after deleting from the wild-type or loxP511 end of insert DNA, respectively. Note that the opposite orientation of the loxP511 site in transposons of the ‘A’ series would have left behind the R (pink) end in a deletion, and this would have interfered during end- sequencing of clones after a second round of deletions with the wild-type loxP transposon. Because both ends of all trans- posons described here share the same 70 nt, primer Seq 1 (20) located within it can sequence the new insert DNA end gen- erated by all of them. However, Seq 1 loses its uniqueness after deletions are made from the other end. Primers unique to each end of the transposons described here are listed in Materi- als and Methods. Although both orientations of the loxP511 site and the RSV neomycin gene cassette were constructed in transposons, and analyzed, only those conforming to the desired uniqueness of sequencing primers are presented in Figure 5 for clarity. The 600 base reads obtained from each deletion-end could BLAST to sequences in the database at the 95–99% homology level. The sequential order of bands in FIGE of the NotI digested DNA from deletion clones corresponded with the order of sequence homology on the chromosome, and BLASTed to only one strand of DNA. DISCUSSION Functionally localizing gene regulatory sequences that operate over large distances using BAC transgenics requires proced- ures to systematically delete DNA at either end of inserts. Both gap-deletions as well as truncations from one end of the reporter gene-tagged BAC insert have been made either by a targeted deletion strategy using homologous recombina- tion, or using loxP transposons (2–5). We believe the loxP transposon deletion strategy has several advantages over homology-based targeted deletion methods. Induction of recombination often triggers intra-insert DNA rearrangements outside of the regions of homology actually targeted: mammalian DNA is known to be recombinogenic, and is