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

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 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.

Figure 1.

Schematic representation of the workflow created to automate the identification of cryptic loxP sites.

Figure 2.

Mouse genome-wide search for cryptic loxP sites. Each graph shows the distribution and the number of cryptic loxP sites in 1 Mb regions along the 21 mouse chromosomes. The length of each graph is proportional to the corresponding chromosomal length. A megabase scale is present at the bottom of each graph.

Figure 3.

(A) Positions of the cryptic loxP sites on the PAC111L11 insert. (B) Sequence comparison between the loxP consensus site and PAC111L11 cryptic loxP sites (primary c-loxP and secondary sc-loxP). Underlined nucleotides — Cre contact points on loxP consensus sequence; bold — conserved nucleotides.

Figure 4.

Growth of PAC111L11-transformed EL350 (A), DY380 (B) and EL250 (C) cells on LB agar supplemented with either 0.2% glucose or 0.2% arabinose. Arrows indicate very small and barely detectable colonies formed on arabinose-containing agar by EL350/PAC111L11.

Figure 5.

Growth of PAC111L11-transformed EL350 on LB agar supplemented with increasing concentrations of glucose. Inset: Un-transformed EL350 and DY380 on agar with 0.2% arabinose.

Figure 6.

(A) pROSA26/Tet plasmid map. (B) Summary of the transformation of pROSA26/Tet plasmid into EL350. (C) PCR analysis of pROSA26/Tet transformed EL350 and DY380 under arabinose-induced and un-induced conditions using T3 and T7 primers. M1: 1 Kb Marker (New England Biolabs). M2: pBluescriptII SK+ plasmid digested with Sau3AI. The 1.5 Kb band indicates the presence of the Tet gene; the 0.2 Kb band is generated after Cre/loxP-mediated excision of the Tet gene.

Figure 7.

In vitro analysis of the presence of nicks in PAC111L11 insert. (A) Schematic representation of expected fragment size for DNA nicked at cryptic loxP1 site. The arrow indicates the location of the c-loxP1 site on the HindIII fragment. P1: probe 1. (B) Schematic representation of predicted fragment sizes for DNA nicked at the c-loxP2 and secondary sc-loxP sites on the EcoRVV fragment. Arrows indicate the location of the c-loxP2 and sc-loxP sites, P2 — probe 2, and P3 — probe 3. (C) Southern analysis of PAC111L11 DNA, following overnight incubation with Cre recombinase, digested with HindIII and hybridized to probe P1 to detect nicks produced at the c-loxP1 site. M:1 kb DNA ladder (NEB) Plus and minus signs on each lane refer to the presence or absence of Cre recombinase and its relative abundance. (D) and (E) Southern analysis of PAC111L11 DNA, following overnight incubation with Cre recombinase, digested with EcoRVV and hybridized with probe P2 (D) and P3 (E) to detect nicks produced by cryptic loxP site 2 and/or sc-loxP. M:1 kb DNA ladder (NEB) Plus and minus signs on each lane refer to the presence or absence of Cre recombinase and its relative concentration.