ABSTRACT
Recently, a codon improved version of the Flpe site specific recombinase, termed Flpo, was reported as having greatly improved performance in mammalian cell applications. However, the degree of improvement could not be estimated because essentially no Flpe activity was observed. Here, we compare Flpe and Flpo accurately in a mammalian cell assay to estimate that Flpo is about five times more active than Flpe and similar to Cre and Dre. Consequently, we generated a Flpo deleter mouse line from the JM8 C57Bl/6 ES cells used in the EUCOMM and KOMP systematic knock-out programs. In breeding experiments, we show that the Flpo deleter delivers complete recombination using alleles that are incompletely recombined by a commonly used Flpe deleter. This indicates that the Flpo deleter is more efficient.
Subject(s)
DNA Nucleotidyltransferases/metabolism , Embryonic Stem Cells/metabolism , Fibroblasts/metabolism , Animals , Base Sequence , Binding Sites/genetics , Cell Line , Cells, Cultured , DNA Nucleotidyltransferases/genetics , Embryo, Mammalian/cytology , Embryo, Mammalian/metabolism , Embryonic Stem Cells/cytology , Female , Fibroblasts/cytology , Gene Expression Regulation, Developmental , Gene Knockout Techniques , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/metabolism , Isoenzymes/genetics , Isoenzymes/metabolism , Male , Mice , Mice, Inbred C57BL , Mice, Transgenic , Plasmids/genetics , Recombination, Genetic , TransfectionABSTRACT
Tyrosine site-specific recombinases (SSRs) including Cre and FLP are essential tools for DNA and genome engineering. Cre has long been recognized as the best SSR for genome engineering, particularly in mice. Obtaining another SSR that is as good as Cre will be a valuable addition to the genomic toolbox. To this end, we have developed and validated reagents for the Dre-rox system. These include an Escherichia coli-inducible expression vector based on the temperature-sensitive pSC101 plasmid, a mammalian expression vector based on the CAGGs promoter, a rox-lacZ reporter embryonic stem (ES) cell line based on targeting at the Rosa26 locus, the accompanying Rosa26-rox reporter mouse line, and a CAGGs-Dre deleter mouse line. We also show that a Dre-progesterone receptor shows good ligand-responsive induction properties. Furthermore, we show that there is no crossover recombination between Cre-rox or Dre-loxP. Hence, we add another set of efficient tools to the genomic toolbox, which will enable the development of more sophisticated mouse models for the analysis of gene function and disease.