Cryptic loxP sites in mammalian genomes: Genome-wide distribution and relevance for the efficiency of BAC/PAC recombineering techniques

S. Semprini, T. J. Troup, N. Kotelevtseva, K. King, J. R E Davis, L. J. Mullins, K. E. Chapman, D. R. Dunbar, J. J. Mullins

    Research output: Contribution to journalArticlepeer-review

    Abstract

    Cre is widely used for DNA tailoring and, in combination with recombineering techniques, to modify BAC/PAC sequences for generating transgenic animals. However, mammalian genomes contain recombinase recognition sites (cryptic lox P sites) that can promote illegitimate DNA recombination and damage when cells express the Cre recombinase gene. We have created a new bioinformatic tool, FuzznucComparator, which searches for cryptic lox P sites and we have applied it to the analysis of the whole mouse genome. We found that cryptic lox P sites occur frequently and are homogeneously distributed in the genome. Given the mammalian nature of BAC/PAC genomic inserts, we hypothesised that the presence of cryptic lox P sites may affect the ability to grow and modify BAC and PAC clones in E. coli expressing Cre recombinase. We have observed a defect in bacterial growth when some BACs and PACs were transformed into EL350, a DH10B-derived bacterial strain that expresses Cre recombinase under the control of an arabinose-inducible promoter. In this study, we have demonstrated that Cre recombinase expression is leaky in un-induced EL350 cells and that some BAC/PAC sequences contain cryptic lox P sites, which are active and mediate the introduction of single-strand nicks in BAC/PAC genomic inserts. © 2007 Oxford University Press.
    Original languageEnglish
    Pages (from-to)1402-1410
    Number of pages8
    JournalNucleic acids research.
    Volume35
    Issue number5
    DOIs
    Publication statusPublished - Mar 2007

    Keywords

    • Animals
    • Attachment Sites, Microbiological
    • Chromosomes, Artificial, Bacterial
    • Chromosomes, Artificial, P1 Bacteriophage
    • Computational Biology
    • genetics: Escherichia coli
    • methods: Genetic Engineering
    • methods: Genomics
    • Humans
    • metabolism: Integrases
    • Mice
    • Recombination, Genetic
    • Software
    • Transformation, Bacterial

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