Dre recombinase, like Cre, is a highly efficient site-specific recombinase in E. coli, mammalian cells and mice

Anastassiadis et al. (2009). Dis Model Mech DOI: 10.1242/dmm.003087 Citations: 330

Cre-loxP was the only recombinase efficient enough for mouse genome engineering. This paper validated Dre and its rox site in bacteria, ES cells, and mice. Dre and Cre showed no cross-recombination, letting both be combined to delete separate exons in one allele.

Parts used

Dre

Key findings

  • Dre recombinase cloned from bacteriophage D6 achieved complete recombination between rox sites in E. coli under arabinose-inducible BAD promoter control with no background activity when uninduced. The temperature-sensitive pSC101 expression system provided excellent on-off regulation that was previously unachievable with Cre constructs. Orthogonality tests confirmed Dre produced no recombination between loxP sites and Cre produced no recombination between rox sites, validating independence from the Cre-loxP system.
  • The researchers generated CAGGs-Dre-IRES-puro constructs and established single-copy integration ES cell lines with germline transmission after 8-cell embryo injection. Rosa26-rox reporter ES cells and accompanying reporter mouse lines were created by gene targeting, providing standardized tools for Dre-mediated recombination. A CAGGs-Dre deleter mouse line was established for ubiquitous expression, and Dre-progesterone receptor fusion demonstrated ligand-responsive induction similar to Cre-ER systems.
  • In vivo experiments with Rosa26-rox reporter mice crossed to CAGGs-Dre deleter mice confirmed Dre recombinase functions with Cre-like efficiency in mammalian genome engineering applications. No crossover recombination occurred between Cre-rox or Dre-loxP combinations, establishing Dre as a fully orthogonal third recombinase system alongside Cre-loxP and Flp-FRT for sophisticated combinatorial site-specific recombination strategies in mice.

Abstract

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.