Abstract
Site-specific recombinases (SSR) are utilized as important genome engineering tools to precisely modify the genome of mice and other model organisms. Reporter mice that mark cells that at any given time had expressed the enzyme are frequently used for lineage tracing and to characterize newly generated mice expressing a recombinase from a chosen promoter. With increasing sophistication of genome alteration strategies, the demand for novel SSR systems that efficiently and specifically recombine their targets is rising and several SSR-systems are now used in combination to address complex biological questions in vivo. Generation of reporter mice for each one of these recombinases is cumbersome and increases the number of mouse lines that need to be maintained in animal facilities. Here we present a multi-reporter mouse line for loci-of-recombination (X) (MuX) that streamlines the characterization of mice expressing prominent recombinases. MuX mice constitutively express nuclear green fluorescent protein after recombination by either Cre, Flp, Dre or Vika recombinase, rationalizing the number of animal lines that need to be maintained. We also pioneer the use of the Vika/vox system in mice, illustrating its high efficacy and specificity, thereby facilitating future designs of sophisticated recombinase-based in vivo genome engineering strategies.
Highlights
Site-specific recombinases are instrumental genetic tools used in a variety of applications, including synthetic biology[1], conditional mutagenesis[2] and lineage tracing[3]
The Vika/vox system has demonstrated to function with high activity in bacteria[25,30], yeast[34] and in mammalian tissue culture cells[30,31] and importantly, no cross-recombination with other Site-specific recombinases (SSR)-systems, or cytotoxicity upon overexpression in mammalian cells has been observed[30]
SSRs have become an integral part in the genetic toolbox of biologists and biomedical scientists
Summary
Site-specific recombinases are instrumental genetic tools used in a variety of applications, including synthetic biology[1], conditional mutagenesis[2] and lineage tracing[3]. The tyrosine recombinase Cre/loxP system is widely utilized in mammalian model organisms, due to its ease of use and efficacy/specificity to recombine its 34 bp loxP-target in vivo. The tyrosine recombinase Flp from the yeast Saccharomyces cerevisiae 2 μ plasmid recombines 34 bp FRT target sites and was discovered around the same time as the Cre/loxP system[11]. Dre has considerable homology to Cre and its target site, rox, only differs to loxP in three out of thirteen nucleotides per half-site[21] Despite this high homology, cross-recombination between the two systems www.nature.com/scientificreports/. Several additional Flp-like[27,28] and Cre-like[29,30,31], recombination systems have been discovered, but none of these systems has been tested for their efficacy and specificity in transgenic mice, yet
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