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.
Semprini
S
S
Molecular Physiology Group, University of Edinburgh Medical School, Edinburgh EH16 4TJ, UK.
Troup
T J
TJ
Kotelevtseva
N
N
King
K
K
Davis
J R E
JR
Mullins
L J
LJ
Chapman
K E
KE
Dunbar
D R
DR
Mullins
J J
JJ
D000818:Animals; D001287:Attachment Sites, Microbiological; D022202:Chromosomes, Artificial, Bacterial; D027042:Chromosomes, Artificial, P1 Bacteriophage; D019295:Computational Biology; D004926:Escherichia coli / Q000235:genetics / Q000254:growth & development; D005818:Genetic Engineering / Q000379:methods*; D023281:Genomics / Q000379:methods*; D006801:Humans; D019426:Integrases / Q000378:metabolism*; D051379:Mice; D011995:Recombination, Genetic; D012984:Software; D014169:Transformation, Bacterial
C045073:Cre recombinase; D019426:Integrases