BMC Genomics 2006, 7:73 http://www.biomedcentral.com/1471-2164/7/73 Table 2: Oligonucleotide sequences (LE, RE) and primers used in the high-throughput LE/RE-spacer mutant loxP screen Name LE LE_EcoRI_Forward LE_HindIII_Reverse RE RE_NotI_Forward RE_NotI_Reverse 1,2,3,6,7,8. They discovered four spacers with vigorous homologous recombination and limited recombination with the wild-type sequence (m2, m3, m7, m11). Only the m2 mutant was tested and found to be functional for RMCE in bacteria and Cre-expressing eukaryotic cells (HEK-293). Other studies have evaluated the in vivo heter- ospecificity of the wild-type spacer versus other published loxP sequences. Siegel et al. tested heterospecific recombi- nation between five published loxP spacer mutants and found that wild-type/FAS, wild-type/2272 and 2272/5171 pairings were incompatible while limited compatibility was found between wild-type/511 sites [18]. However, another study found equal recombination efficiency into a BAC between the wild-type and lox511 spacers [19]. Recently, a phage P1 transduction assay recapitulated the sensitivity and exclusivity of the lox2272 and lox5171 spacer mutants [20]. However, to the best of our knowl- edge, no studies have investigated if the wild-type spacer is the most efficient sequence for Cre-mediated recombi- nation. Given that spacer and inverted repeat mutants have been used together successfully, it may be possible to introduce numerous DNA segments into a given target molecule, chromosome or genome if a sufficient number of non- promiscuous LE/RE-spacer mutants can be identified. In the present study we have developed and executed a high- throughput in vitro strategy to rapidly identify novel LE/ RE-spacer mutants and determine their specificity. Results The recombination reaction Two oligonucleotides (LE, RE) were designed that con- tained loxP sites with six degenerate spacer nucleotides (positions 1,2,3,6,7,8) and two central fixed spacer nucle- otides (4 and 5) (Figure 1, Table 2). The central nucle- otides in the 4, 5 position were limited to thymine and adenine residues because these have been previously sug- gested by mutational analysis to be essential for strand exchange [6]. One of the oligonucleotide pools (LE) con- tained the lox71 left arm mutant sequence and the other Sequence 5' < CTGAGCTCGGGTGCCTGTTACCGTTCGTATANNNTANNNTAT ACGAAGTTATGACTGCACCCATCAGAGC > 3' 5' < TGACGAATTCCTGAGCTCGGGTGCCTGT > 3' 5' < GCTAAAGCTTGCTCTGATGGGTGCAGTC > 3' 5' < TGTCTCTGGCACGCTAGGATAACTTCGTATANNNTANNNTAT ACGAACGGTATATGGCCTTCTGTAGCGG > 3' 5' < GTACGCGGCCGCTGTCTCTGGCACGCTAGG > 3' 5' < GTACGCGGCCGCCCGCTACAGAAGGCCATA > 3' (RE) contained the lox66 right arm mutant sequence (Fig- ure 1, Table 2) [12]. These oligonucleotides were con- verted to double stranded product by PCR. Primers used to generate the LE PCR product were tailed with EcoRI and HindIII restriction sites for subcloning into the pUC19 vector and primers used to generate the RE PCR product were tailed with NotI restriction sites (Table 2). These two pools of oligonucleotides were then incubated in the pres- ence of Cre recombinase (Figure 1). Sequencing and analyzing the successful recombinants The desired 1.8 kbp recombination products were ampli- fied by PCR using the RE_NotI forward and reverse prim- ers (Table 2) then digested with NotI, agarose gel purified and re-circularized with T4 DNA ligase to generate a library of paired loxP recombination products in pUC19 vector (Figure 1). These plasmids were transformed into DH10B cells, grown overnight, and plated on solid media. Each individual colony (clone) represented a distinct, suc- cessful, recombination reaction between two loxP spacer sequences. A total of 5,670 clones were picked, grown overnight, and plasmid DNA was isolated and sequenced with M13 Reverse sequencing primer using previously described methods (Figure 1) [21]. Of these clones, 4,992 yielded successful sequence (See Additional files 1 &2). The M13 Reverse sequencing primer generated data from the negative strand (Figure 1, Figure 2). We sequenced off of the negative strand because the position of the M13 Forward primer was too close to the lox71/lox66 double mutant to reliably generate the sequence of both spacers. A limited number of clones (768) were sequenced in the positive orientation but only 42/768 produced high qual- ity spacer sequences (See Additional files 1 &2) all of which agreed with that of the negative strand. According to the reaction mechanism, we expected to see wild-type inverted repeats flanking one spacer and the lox66 and lox71 inverted repeats flanking the other spacer post-recombination (Figure 3) [12,13]. Consequently, a typical sequencing read was composed of the following Page 3 of 13 (page number not for citation purposes)