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

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 loxP 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 loxP sites and we have applied it to the analysis of the whole mouse genome. We found that cryptic loxP 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 loxP 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 loxP sites, which are active and mediate the introduction of single-strand nicks in BAC/PAC genomic inserts

Schematic representation of the workflow created to automate the identification of cryptic P sites

<p><b>Copyright information:</b></p><p>Taken from "Cryptic P sites in mammalian genomes: genome-wide distribution and relevance for the efficiency of BAC/PAC recombineering techniques"</p><p></p><p>Nucleic Acids Research 2007;35(5):1402-1410.</p><p>Published online 6 Feb 2007</p><p>PMCID:PMC1865043.</p><p>© 2007 The Author(s).</p

Growth of PAC111L11-transformed EL350 (), DY380 () and EL250 () cells on LB agar supplemented with either 0

<p><b>Copyright information:</b></p><p>Taken from "Cryptic P sites in mammalian genomes: genome-wide distribution and relevance for the efficiency of BAC/PAC recombineering techniques"</p><p></p><p>Nucleic Acids Research 2007;35(5):1402-1410.</p><p>Published online 6 Feb 2007</p><p>PMCID:PMC1865043.</p><p>© 2007 The Author(s).</p>2% glucose or 0.2% arabinose. Arrows indicate very small and barely detectable colonies formed on arabinose-containing agar by EL350/PAC111L11

Growth of PAC111L11-transformed EL350 on LB agar supplemented with increasing concentrations of glucose

<p><b>Copyright information:</b></p><p>Taken from "Cryptic P sites in mammalian genomes: genome-wide distribution and relevance for the efficiency of BAC/PAC recombineering techniques"</p><p></p><p>Nucleic Acids Research 2007;35(5):1402-1410.</p><p>Published online 6 Feb 2007</p><p>PMCID:PMC1865043.</p><p>© 2007 The Author(s).</p> Inset: Un-transformed EL350 and DY380 on agar with 0.2% arabinose

Australian square kilometre array pathfinder : I. system description

In this paper, we describe the system design and capabilities of the Australian Square Kilometre Array Pathfinder (ASKAP) radio telescope at the conclusion of its construction project and commencement of science operations. ASKAP is one of the first radio telescopes to deploy phased array feed (PAF) technology on a large scale, giving it an instantaneous field of view that covers 31 deg(2) at 800MHz. As a two-dimensional array of 36x12 m antennas, with baselines ranging from 22 m to 6 km, ASKAP also has excellent snapshot imaging capability and 10 arcsec resolution. This, combined with 288 MHz of instantaneous bandwidth and a unique third axis of rotation on each antenna, gives ASKAP the capability to create high dynamic range images of large sky areas very quickly. It is an excellent telescope for surveys between 700 and 1800MHz and is expected to facilitate great advances in our understanding of galaxy formation, cosmology, and radio transients while opening new parameter space for discovery of the unknown