BMC Genomics 2006, 7:73 Table 1: Published mutant loxP sites Wild-type lox 511 [6] lox 5171 [7] lox 2272 [7] m2 [8] m3 [8] m7 [8] m11 [8] lox 71 [12, 13] lox 66 [12, 13] loxP site Left inverted repeat sequence Spacer (5'→3') ATAACTTCGTATA ATAACTTCGTATA ATAACTTCGTATA ATAACTTCGTATA ATAACTTCGTATA ATAACTTCGTATA ATAACTTCGTATA ATAACTTCGTATA taccgTTCGTATA ATAACTTCGTATA ATGTATGC ATGTATaC ATGTgTaC AaGTATcC AgaaAcca taaTAcca AgaTAgaa cgaTAcca ATGTATGC ATGTATGC http://www.biomedcentral.com/1471-2164/7/73 Right inverted repeat sequence TATACGAAGTTAT TATACGAAGTTAT TATACGAAGTTAT TATACGAAGTTAT TATACGAAGTTAT TATACGAAGTTAT TATACGAAGTTAT TATACGAAGTTAT TATACGAAGTTAT TATACGAAcggta Background Cre-loxP recombination is an important tool in molecular genetics. Cre ("Causes recombination") recombinase from bacteriophage P1 recognizes a specific 34 bp target sequence, termed loxP, composed of an 8 bp spacer region flanked by two identical 13 bp inverted repeats [1] (Table 1). Each base in the spacer region is conventionally named 1,2,3,4,5,6,7,8 according to its order in the sequence. Cre-loxP sites mediate site specific intra- or inter-strand exchange of DNA molecules catalyzed by Cre recombinase. Depending on the location and the orienta- tion of these sites, they can invert, insert, delete or exchange fragments of DNA in prokaryotic or eukaryotic systems [2-5]. Orientation of insert DNA post-recombina- tion is dependent on the orientation of the sites prior to the reaction, with sites in the same orientation on a given DNA strand mediating excision of intervening sequence and sites in opposite orientation mediating inversion of intervening sequence. Since the excision reaction is kinet- ically favoured over the insertion reaction, gene deletion/ inactivation experiments are straightforward to engineer by flanking the target sequence with loxP sites. The diffi- culty in accomplishing DNA insertion is that the insertion reaction results in the presence of two loxP sites in cis post- recombination, which themselves become substrates for Cre and lead to rapid excision of the inserted segment. Two classes of variant loxP sites are available to engineer stable Cre-loxP integrative recombination. Both exploit sequence mutations in the Cre recognition sequence, either within the 8 bp spacer region or the 13-bp inverted repeats. Spacer mutants such as lox511[6], lox5171 [7], lox2272 [7], m2, m3, m7, and m11 [8] (Table 1) recombine readily with themselves but have a markedly reduced rate of recombination with the wild-type site. This class of mutants has been exploited for DNA insertion by Recom- binase Mediated Cassette Exchange (RMCE) [9-11]. Inverted repeat mutants represent the second class availa- ble and contain altered bases in the left inverted repeat (LE mutant) or the right inverted repeat (RE mutant). The LE mutant, lox71, has 5 bp on the 5' end of the left inverted repeat that are changed from the wild type sequence to TACCG [12,13] (Table 1). Similarly, the RE mutant, lox66, has the five 3'-most bases changed to CGGTA (Table 1). Inverted repeat mutants are used for integrating plasmid inserts into chromosomal DNA with the LE mutant designated as the "target" chromosomal loxP site into which the "donor" RE mutant recombines. Post-recombination, loxP sites are located in cis, flanking the inserted segment. The mechanism of recombination is such that post-recombination one loxP site is a double mutant (containing both the LE and RE inverted repeat mutations) and the other is wild type [14-16]. The double mutant is sufficiently different from the wild-type site that it is unrecognized by Cre recombinase and the inserted segment is not excised. Recently, spacer and inverted repeat mutants have been combined to increase the spe- cificity and stability of integrative recombination [8,17]. In past experiments, novel spacer mutants were discov- ered by mutating a suite of bases [6,7] or generating a set of potential spacer mutants and testing recombination between these spacers with the wild-type loxP site [8]. Lee and Saito used an in vitro assay that evaluated the recom- bination efficiency of 24 spacers with 1 bp substitutions and 30 spacers with 2 bp substitutions from the sequence of the wild-type loxP [7]. Their data suggested that homol- ogy was required at positions 2–5 and positions 6–7 for efficient strand exchange and resolution of the Holiday junction whereas positions 1 and 8 had relaxed homology requirements. They concluded that homology was essen- tial to achieve recombination rates between mutant loxP spacers comparable to that of the wild-type sequence. Their success with the lox2272 mutant suggested that posi- tions 2 and 7 were particularly important in blocking pro- miscuous recombination. Langer et al. focused on the in vivo functionality of novel loxP sites in bacterial and eukaryotic systems [8]. In their work, a suite of random loxP sites were generated by utilizing a template loxP oli- gonucleotide with degenerate spacers at positions Page 2 of 13 (page number not for citation purposes)