We are analyzing https://link.springer.com/article/10.1186/1471-2164-7-73.

Title:
A high-throughput screen identifying sequence and promiscuity characteristics of the loxP spacer region in Cre-mediated recombination | BMC Genomics
Description:
Background Cre-loxP recombination refers to the process of site-specific recombination mediated by two loxP sequences and the Cre recombinase protein. Transgenic experiments exploit integrative recombination, where a donor plasmid carrying a loxP site and DNA of interest integrate into a recipient loxP site in a target genome. Unfortunately, integrative recombination is highly inefficient because the insert is flanked by two loxP sites, which themselves become targets for Cre and lead to subsequent excision of the insert. A small number of mutations have been discovered in parts of the loxP sequence, specifically the spacer and inverted repeat segments, that increase the efficiency of integrative recombination. In this study we introduce a high-throughput in vitro assay to rapidly detect novel loxP spacer mutants and describe the sequence characteristics of successful recombinants. Results We created synthetic loxP oligonucleotides that contained a combination of inverted repeat mutations (the lox 66 and lox 71 mutations) and mutant spacer sequences, degenerate at 6 of the 8 positions. After in vitro Cre recombination, 3,124 recombinant clones were identified by sequencing. Included in this set were 31 unique, novel, self-recombining sequences. Using network visualization tools, we recognized 12 spacer sets with restricted promiscuity. We observed that increased guanine content at all spacer positions save for position 8 resulted in increased recombination. Interestingly, recombination between identical spacers was not preferred over non-identical spacers. We also identified a set of 16 pairs of loxP spacers that reacted at least twice with another spacer, but not themselves. Further, neither the wild-type P1 phage loxP sequence nor any of the known loxP spacer mutants appeared to be kinetically favoured by Cre recombinase. Conclusion This study approached loxP spacer mutant screening in an unbiased manner, assuming nothing about candidate 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 subset of loxP sites identified here are candidates for in vivo serial recombination as they have already demonstrated limited promiscuity with other loxP spacer and stability in the presence of Cre.
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Keywords {🔍}

spacer, recombination, sequence, loxp, spacers, sites, figure, sequences, inverted, pubmed, article, mutant, lox, cre, mutants, oligonucleotides, positions, wildtype, type, reactions, input, repeat, file, table, scholar, google, recombinase, dna, cas, site, position, reaction, strand, data, products, promiscuity, nonself, analysis, successful, central, additional, selfrecombining, pcr, mismatches, number, creloxp, original, published, full, sequencing,

Topics {✒️}

high-fidelity bac/pac retrofitting open access article cre-loxp site-specific recombination gene deletion/inactivation experiments article download pdf health research scholar full size image multiplexed site-specific recombination p1 site-specific recombination site-specific dna recombination site-specific recombination mediated future cre-loxp applications wild-type inverted repeat cre-loxp spacer crosstalk double-reciprocal crossover mediated wild-type inverted repeats cre-expressing eukaryotic cells vigorous homologous recombination subsequent cre-mediated recombination wild-type spacer versus cre-loxp site promiscuity homologous identical sequences identify spacer cross-talk wild-type spacer sequence left inverted repeat cre gene mediated full access related subjects central ta nucleotides insert dna post-recombination inverted repeat mutant tyrosine recombinases privacy choices/manage cookies inverted repeat segments cre-loxp recombination transgenic dna segments loxp recombination site high throughput sequencing supercoiled le/puc19 plasmid vivo phagemid system multiple fasta file wild-type loxp [7] inverted repeat mutants cre-mediated recombination inverted repeat mutations recipient loxp site site-specific integration loxp site determines m13 reverse primer arm mutant sequence

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         headline:A high-throughput screen identifying sequence and promiscuity characteristics of the loxP spacer region in Cre-mediated recombination
         description:Cre-loxP recombination refers to the process of site-specific recombination mediated by two loxP sequences and the Cre recombinase protein. Transgenic experiments exploit integrative recombination, where a donor plasmid carrying a loxP site and DNA of interest integrate into a recipient loxP site in a target genome. Unfortunately, integrative recombination is highly inefficient because the insert is flanked by two loxP sites, which themselves become targets for Cre and lead to subsequent excision of the insert. A small number of mutations have been discovered in parts of the loxP sequence, specifically the spacer and inverted repeat segments, that increase the efficiency of integrative recombination. In this study we introduce a high-throughput in vitro assay to rapidly detect novel loxP spacer mutants and describe the sequence characteristics of successful recombinants. We created synthetic loxP oligonucleotides that contained a combination of inverted repeat mutations (the lox 66 and lox 71 mutations) and mutant spacer sequences, degenerate at 6 of the 8 positions. After in vitro Cre recombination, 3,124 recombinant clones were identified by sequencing. Included in this set were 31 unique, novel, self-recombining sequences. Using network visualization tools, we recognized 12 spacer sets with restricted promiscuity. We observed that increased guanine content at all spacer positions save for position 8 resulted in increased recombination. Interestingly, recombination between identical spacers was not preferred over non-identical spacers. We also identified a set of 16 pairs of loxP spacers that reacted at least twice with another spacer, but not themselves. Further, neither the wild-type P1 phage loxP sequence nor any of the known loxP spacer mutants appeared to be kinetically favoured by Cre recombinase. This study approached loxP spacer mutant screening in an unbiased manner, assuming nothing about candidate 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 subset of loxP sites identified here are candidates for in vivo serial recombination as they have already demonstrated limited promiscuity with other loxP spacer and stability in the presence of Cre.
         datePublished:2006-04-04T00:00:00Z
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            Proteomics
            Animal Genetics and Genomics
            Microbial Genetics and Genomics
            Plant Genetics and Genomics
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      headline:A high-throughput screen identifying sequence and promiscuity characteristics of the loxP spacer region in Cre-mediated recombination
      description:Cre-loxP recombination refers to the process of site-specific recombination mediated by two loxP sequences and the Cre recombinase protein. Transgenic experiments exploit integrative recombination, where a donor plasmid carrying a loxP site and DNA of interest integrate into a recipient loxP site in a target genome. Unfortunately, integrative recombination is highly inefficient because the insert is flanked by two loxP sites, which themselves become targets for Cre and lead to subsequent excision of the insert. A small number of mutations have been discovered in parts of the loxP sequence, specifically the spacer and inverted repeat segments, that increase the efficiency of integrative recombination. In this study we introduce a high-throughput in vitro assay to rapidly detect novel loxP spacer mutants and describe the sequence characteristics of successful recombinants. We created synthetic loxP oligonucleotides that contained a combination of inverted repeat mutations (the lox 66 and lox 71 mutations) and mutant spacer sequences, degenerate at 6 of the 8 positions. After in vitro Cre recombination, 3,124 recombinant clones were identified by sequencing. Included in this set were 31 unique, novel, self-recombining sequences. Using network visualization tools, we recognized 12 spacer sets with restricted promiscuity. We observed that increased guanine content at all spacer positions save for position 8 resulted in increased recombination. Interestingly, recombination between identical spacers was not preferred over non-identical spacers. We also identified a set of 16 pairs of loxP spacers that reacted at least twice with another spacer, but not themselves. Further, neither the wild-type P1 phage loxP sequence nor any of the known loxP spacer mutants appeared to be kinetically favoured by Cre recombinase. This study approached loxP spacer mutant screening in an unbiased manner, assuming nothing about candidate 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 subset of loxP sites identified here are candidates for in vivo serial recombination as they have already demonstrated limited promiscuity with other loxP spacer and stability in the presence of Cre.
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      dateModified:2006-04-04T00:00:00Z
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         Space Sequence
         Inverted Repeat
         loxP Site
         Recombination Reaction
         loxP Sequence
         Life Sciences
         general
         Microarrays
         Proteomics
         Animal Genetics and Genomics
         Microbial Genetics and Genomics
         Plant Genetics and Genomics
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