(A) Schematic diagram of the integration scheme. The donor vector containing a DOI integrates into a genomically tagged site (Rosa26 in this figure) in the targeted allele by Cre-based RMCE via mutant loxP sites. Extra sequence in the RMCE^ex allele is removed by FLPe, resulting in an RMCE^Δex allele. Red and blue, and the green arrowheads are the PCR primer sets used for detection of correct recombinant (B). (B) Targeted insertion of mutant loxP-tagged sequences into the H2-Tw3 and Rosa26 loci. H2-Tw3 and Rosa26 targeting vectors were introduced into E14tg2a ES cells. G418-resistant clones were picked, and the targeted alleles were screened by PCR and certified by hybridization. Southern blot analyses of genomic DNA from ES cells (H2-Tw3) and mice (Rosa26) are shown on the right side of the figure. Homologous recombinations at the H2-Tw3 and Rosa26 loci are detectable using BglI or BamHI-digested genomic DNAs by the probes marked by the red bars, respectively.

Efficient detection of correct RMCE recombinants in ES cells, and EGFP expression in RMCE^ex and RMCE^Δex alleles. (A) The rate of appearance of the background clones. Donor vector, which contains lacZ gene as promoterless reporter gene (A), was introduced into targeted ES cells with or without Cre-expression plasmid. Only 0.5–1.0% of colonies regarded as non-recombined clone stained blue with X-gal after introducing donor vector alone, whereas 20.6–24.3% of colonies showed lacZ expression upon co-introduction of Cre plasmid. (B) Efficient detection of correctly recombined clones. Introduction of a donor vector, in which EGFP gene was included as promoterless reporter gene, into ES cells together with the Cre plasmid resulted in the appearance of hygromycin-resistant EGFP-fluorescent and -non-fluorescent clones. Among them, 48 green fluorescent colonies (24 from H2-Tw3 and 24 from Rosa26) were picked and analyzed by PCR. All the collected clones showed correct recombination (100%; Supplementary Table S2). (C) EGFP expression in ES cells (left) and intestine of the mouse (right) containing a single copy of the ‘CAG-EGFP-polyA’ cassette at the H2-Tw3 and Rosa26 loci. EGFP expression of both ES cells and intestines with RMCE^ex allele was silenced (weak and mosaic). In the RMCE^Δex allele that the vector backbone and the bacterial resistance gene were removed by FLPe administration, ES cells and intestines exhibited robust EGFP fluorescence.

PITT system. (A) PITT efficiency. A total of 11 constructs were microinjected into fertilized eggs containing the targeted allele. The numbers of founder transgenic mice obtained by PITT are shown in red. (B) PCR screening of correct recombinants. Primer sets used to amplify the junction regions and internal sequence of the donor vector, are shown in red and blue, and the green arrowheads in A, respectively. Genomic junctions expected for an RMCE and internal regions were amplified in the correct recombinants (Nos. 3 and 16), while only the internal regions were amplified from a pup with random integration (No. 2).

Fluorescent expression in targeted transgenic mice. (A) EGFP fluorescence in H2-Tw3^CAG::EGFP and Rosa26^CAG::EGFP mice, containing a single copy of the ‘CAG-EGFP-polyA’ cassette at the H2-Tw3 and Rosa26 loci, respectively. Three independent transgenic lines were obtained for H2-Tw3^CAG::EGFP: two lines, labeled 2 and 3, were obtained by PITT, while the other, labeled 4, was obtained via RMCE in ES cells. The Rosa26^CAG::EGFP line, labeled 5, was obtained via RMCE in ES cells. (B) Analysis of EGFP fluorescence in spleen cells prepared from the targeted transgenic mice. The numbers indicate mouse lines that correspond to those in (A). (C–E) Expression of fluorescent genes in colored mice. All lines containing a single copy of the ‘CAG-fluorescent gene-polyA’ cassette at the Rosa26 locus exhibited ubiquitous and very bright fluorescence in blastocysts (C), newborn mice (D) and adult mice (E). (F) Ubiquitous and uniform fluorescence expression in tissues of Rosa26^CAG::EGFP mice. The left panels (a–i) are bright-field images, and the right panels (a′–i′) are dark-field fluorescent images taken with green filters. Rosa26^CAG::EGFP (g,g′–i,i′) mice showed generalized pancellular FP expression, while variegated EGFP expression was observed in tissues of the green mouse (d,d′–f,f′). All transgenic mice were hemizygotes for the transgene.

Tyrosinase (Tyr) knockdown mouse. (A) The artificial miRNA for Tyr was expressed under the control of the CAG promoter at the Rosa26 locus (Rosa26^CAG::miR-Tyr). (B) The levels of Tyr mRNA were quantitated by real-time RT-PCR analysis. Coat color (A), melanin in ears (C) and choroid coat (D) were compared between the wild-type and Rosa26^CAG::miR-Tyr mice.