A single reporter mouse line for Vika, Flp, Dre, and Cre-recombination

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.

Figure 1

Vika recombines specifically its target sites (vox) in mES cells. (a) Schematic diagram of the Rosa26-vox-neo-vox-lacZ reporter before and after Vika recombination. Two mESC clones (#3 and #9) with stably integrated lacZ reporters were transiently transfected with CAG-Vika-IRES-puro and analysed at 24 h and 48 h by lacZ staining. (b) Each of the four recombinase expression vectors (3 µg each per 6-well) were transfected in the four stable Rosa26 reporter mES cells lines. Cells were analysed 48 h after transfection by lacZ staining. (Scale bar = 100 µm).

Figure 2

The multi site reporter vector is functional in mESCs. (a) Schematic diagram of the Rosa26-multi site reporter (MuX) before and after Vika, Flpo, Dre and Cre recombination. Stable mESC reporter clones were transiently transfected with expression vectors and analysed at 48 h for GFP expression. (b) Flow cytometry to measure GFP levels after transfecting different amounts of the recombinase expression vectors (0.05 to 1 µg per 6-well). Representative plots of cells transfected with 1 µg recombinase expression vector are shown. (c) Quantification of (b). Error bars represent standard deviations. Statistical significance is shown only for P < 0.01 (**) and P < 0.001 (***).

Figure 3

The MuX reporter is functional in mice. (a) Micrographs showing embryos at E10.5 isolated from intercrosses between Vika, Flpo, Dre and Cre deleter male mice with Rosa26-VFRL-eGFP (MuX) reporter mice. The reporter protein is ubiquitously expressed. Scale bar = 1 mm. (b) Sections of E10.5 embryos. (c) PCR product from each of the four deleter x MuX reporter crosses followed by digestion with indicated restriction endonucleases (− = undigested, N = NheI, As = AscI, P = PvuI, A = AgeI).

Figure 4

Vika-recombination is complete in adult tissues. (a) Organs isolated from MuX × Vika and MuX × Cre mice and analysed for GFP expression. Scale bar = 1 mm. (b) Schematic diagram of the Southern strategy for detecting recombination. (c) Southern blot showing complete recombination in all analysed tissues.