BMC Genomics 2006, 7:73 http://www.biomedcentral.com/1471-2164/7/73 Table 3: Total reactions and mutations per position in the 8 bp loxP spacer region organized by mismatch Type Type Type I Type II Type III Type IV Total Pos 1 0 209 0 1,575 1,784 Pos 2 0 0 144 1,361 1,505 Pos 3 0 0 126 1,104 1,230 Pos 4 Pos 5 0 0 1 3 4 0 0 0 1 1 Pos 6 0 0 167 1,349 1,516 Pos 7 0 0 143 1,331 1,474 Pos 8 0 244 0 2,128 2,372 Total Reactions 47 303 284 2,490 3,124 Thus, each spacer in the library had a promiscuity profile defined by the suite of loxP sites with which it recom- bined. As previously discussed, since Type IV mismatches had ambiguous input oligonucleotides they were not included in this section of the analysis (Figure 4D). Inferred spacer sequences were divided into two sets: self (a spacer sequence that recombined with itself plus one or more other spacer sequences) and non-self (a spacer sequence that did not recombine with itself, but did recombine with another non-self spacer more than once). For non-self recombining pairs, we screened for the subset that were replicated because the majority of spacer pairs were singleton non-self spacer pairs. The self and non-self sets are mutually exclusive. In the set of 3,124 successful recombination reactions, 32 self-recombining spacers were discovered (Figure 7). Of these, only one spacer AGGTATGC or lox23 has been described previously [7], the remaining 31 are novel self-recombining spacers. Spacers TTTTAGGT and GGCTATAG recombined solely with themselves but this exclusivity may be a reflection of limited sampling rather than a property of the spacer. Self- recombination events were correlated with increasing total recombination reactions (r2 = 0.89, p < 0.05). The most promiscuous self-recombining sequence was GGG- TAGGC which underwent 44 insertional recombination reactions. There were also a set of 16 non self-recombin- ing spacers (Table 5). Of these TGGTAGGC and TGG- TAGGT recombined with each other most frequently (4 reactions). The most promiscuous non self-recombining spacer was GGGTAGGA with 26 total reactions. We tested with the χ2 statistic if each spacer from both the self and non-self sets recombined with equal frequency with all of their partners. All spacers did not significantly deviate from the hypothesis of equal recombination fre- quency with all their partners save for GGGTAGGG (χ2, p < 0.05). The spacer GGGTAGGG deviated from the hypothesis because it had higher promiscuity within a number of non-identical spacers rather than with itself (i.e. TGGTAGGC, GGGTAGGT, TGGTAGGA all recom- bined more frequently with GGGTAGGG than GGG- TAGGG did with itself). Selecting candidate spacers Traditionally candidate loxP spacer sequences with the greatest potential utility for genetic engineering will self- recombine and exhibit limited promiscuity. Some pro- miscuity is tolerable if the sites prone to interaction are used in constructs in a mutually exclusive manner. We vis- ualized self and non-self spacer interactions as a network using Cytoscape [25] in order to identify spacer cross-talk and the periphery of promiscuity (Figure 8). The hubs in the middle of this interaction map are likely too promis- cuous to be useful. Based on the interaction map, we iden- tified the following peripheral 11 self and 1 non-self non- promiscuous spacers that had no more than two partners in our library [spacer sequence (# of other self-recombin- ing partners)]: GTATAGTA (0), GGCTATAG (0), TCG- TAGGC (2), GCGTATGT (2), TTGTATGG (1), GGATAGTA (1), GTGTATTT (1), AGGTATGC (1), GGTTACGG (1), TTTTAGGT (1), and GAGTACGC (1) and [GTGTACGC (2) and GTGTACGG (2)] (non-self set). Discussion The present study approaches loxP spacer mutant screen- ing in an unbiased manner, assuming nothing about can- didate loxP sites save for the conserved 4 and 5 spacer positions. Candidate sites were free to recombine with any other sequence in the pool of all possible sites. The present strategy also tested the general promiscuity of loxP spacer sequences. Cre-loxP spacer crosstalk was directly Table 4: Base frequency (%) by position of each input loxP spacer sequence that successfully recombined Pos A: T: G: C: Total 1 8.07% 25.88% 59.60% 6.45% 6,248 2 9.31% 23.40% 55.67% 11.62% 6,248 3 8.98% 11.72% 70.50% 8.80% 6,248 4 0.00% 99.94% 0.00% 0.06% 6,248 5 99.98% 0.02% 0.00% 0.00% 6,248 6 12.36% 20.95% 55.27% 11.43% 6,248 7 11.89% 18.61% 60.68% 8.82% 6,248 8 17.13% 22.25% 28.30% 32.33% 6,248 Total 10,479 13,918 20,619 4,968 49,984 Page 7 of 13 (page number not for citation purposes)