Ectopic Cre activity in a subset of Osr2^IresCre:R26R double heterozygous embryos. (A–C) Three E12.5 Osr2^IresCre:R26R double heterozygous embryos representing the three different X-gal staining patterns. Most embryos analyzed displayed tissue-specific X-gal staining consistent with specific Cre expression from the Osr2 locus, as shown in A. Approximately 20% of the double heterozygous embryos displayed some ectopic X-gal staining, as shown in B. Approximately 10% of the double heterozygous embryos displayed almost ubiquitous X-gal staining, as shown in C.

Detection of Cre-mediated activation of LacZ expression during palate development in the Osr2^IresCre:R26R double heterozygous embryos. (A) At E11.5, palatal outgrowths arise from the oral side of the maxillary processes. Beta-galactosidase activity was detected highly specifically in the nascent palatal mesenchyme (black arrow). (B) Frontal section of an E12.5 Osr2^IresCre:R26R double heterozygous embryo showing tissue-specific X-gal staining in the palatal mesenchyme, and parts of the mandibular mesenchyme. (C) Sagittal section of an E14.5 Osr2^IresCre:R26R double heterozygous embryo showing X-gal staining throughout the anteroposterior axis of the secondary palate. (D) Frontal section of an E15.5 Osr2^IresCre:R26R double heterozygous embryo showing intense X-gal staining in the palatal mesenchyme and in the molar dental mesenchyme. de, dental epithelium; dm, dental mesenchyme; ey, eye; man, mandible; max, maxillary process; nc, nasal cavity; p, palate; ps, palatal shelf; to, tongue.

Whole mount X-gal staining detection of beta-galactosidase expression in Osr2^IresCre:R26R double heterozygous embryos (A,C,E,F) in comparison with that in Osr2^tm1Jian (Osr2-lacZ) heterozygous embryos. (A) At E10.0, beta-galactosidase expression (blue color) was detected in the mesonephric tissues (green arrow) in Osr2^IresCre:R26R double heterozygous embryos. There was also beta-galactosidase expression in the region immediately caudal to the second branchial arches (red arrowhead), which was not observed in Osr2-lacZ embryos (B). (B) At E10.0, beta-galactosidase activity was detected in the mesonephros (green arrow), in the forelimb buds (white arrowhead), and was beginning to be expressed in the rostro-proximal mandibular mesenchyme (black arrow), in the Osr2-lacZ heterozygous embryos. (C) By E10.5, beta-galactosidase activity was detected in the forelimb buds (white arrowhead) and in the rostro-proximal mandibular region (black arrow) in Osr2^IresCre:R26R double heterozygous embryos. (D) At the same developmental stage (E10.5) as the embryo in C, Osr2-lacZ heterozygous embryos exhibit more intense X-gal staining in the rostro-proximal mandibular region (black arrow) as well as expression in the hindlimb buds. (E, F) By E10.75 to E11.0, the Osr2^IresCre:R26R double heterozygous embryos exhibit robust beta-galactosidase expression in the rostroproximal region of the mandibular processes (black arrow) and in the nascent palatal mesenchyme (black arrowheads) next to the oral side of the maxillary processes. ba2, second branchial arch; fl, forelimb bud; hl, hindlimb bud; man, mandibular process; max, maxillary process.

Detection of Cre-mediated activation of beta-galactosidase expression during kidney development in the Osr2^IresCre:R26R double heterozygous embryos. (A) A transverse section through the mesonephros and the rostral cortex of the developing metanephric kidney of an E12.5 Osr2^IresCre:R26R double heterozygous embryo showing intense X-gal staining in the mesonephric tubules. The Woffian duct epithelium is devoid of beta-galactosidase activity whereas the mesenchyme immediately surrounding the Woffian duct shows X-gal staining. (B) A transverse section through the middle of the developing metanephric kidney of an E12.5 Osr2^IresCre:R26R double heterozygous embryo showing highly specific and restricted X-gal staining in the metanephric mesenchyme derived epithelial S-shaped bodies. (C) High magnification view of a region of the section shown in B containing one well-recognizable S-shaped body structure. The X-gal staining is restricted to the developing glomerulus capsule epithelia and is completely absent from the distal tubule connecting to the S-shaped body. The collecting duct epithelia and the kidney stromal mesenchyme are completely devoid of beta-galactosidase activity. (D) A transverse section through the developing metanephric kidney of an E14.5 Osr2^IresCre:R26R double heterozygous embryo showing intense X-gal staining in the maturing glomeruli (black arrows). A subset of the epithelial tubules, most-likely the metanephric mesenchyme derived proximal tubules of the nephrons, were also intensely stained. The collecting duct epithelia and the kidney stromal mesenchyme remain negative for beta-galactosidase activity. cd, collecting duct; dt, distal tubule; gl, glomerulus; gr, genital ridge; ki, kidney; mt, mesonephric tubule; pt, proximal tubule; sb, S-shaped body; wd, Wolffian duct.

Targeted insertion of the IresCre bicistronic expression cassette into the 3′ UTR of the mouse Osr2 gene. (A) The Osr2 gene consists of four exons spanning approximately 8 kb of genomic DNA. Boxes indicate exons, with the protein-coding region marked in black. The positions of the translation start (ATG) and stop (TAG) codons are also indicated. Restriction sites are: B, BamHI; E, EcoRI; H, HindIII; P, PstI; X, XbaI. The targeting vector used the 2.6 kb PstI-XbaI fragment containing sequences from Exon 2 to the 3′ UTR region in Exon 4 as the 5′ arm and the 3.3 kb XbaI-HindIII fragment 3′ to the Osr2 coding region as the 3′ arm. The IresCre cassette and a FRT-flanked neo expression cassette were inserted in between the arms and a diptheria toxin A (DTA) expression cassette was cloned 3′ to the 3′ arm for negative selection against random integration. Correct targeting results in insertion of the IresCre cassette and the neo cassette at the XbaI site in the 3′ UTR. (B) Southern hybridization analysis of selected embryonic stem cell DNA samples to confirm correct targeting. Genomic DNA samples were digested with BamHI, separated by electrophoresis through a 1% agarose gel, transferred onto a GeneScreen Plus nylon membrane (PerkinElmer), and hybridized with random prime-labeled probes made from the 600 bp HindIII-EcoRI fragment isolated from the Intron 1 region 5′ to the targeted region. The 14 kb BamHI fragment corresponding to the wild-type allele was detected in all ES DNA samples, whereas the 7.2 kb fragment was only detectable in the DNA samples from correctly targeted ES clones. (C) PCR analysis of tail DNA samples from a litter of newborn F2 progeny. The fragments amplified from wild-type and Osr2^IresCre alleles are 490 bp and 706 bp, respectively. Homozygous mutants were born at the expected Mendelian frequency and lived normally. +/+, wild-type; +/−, heterozygote; −/−, homozygote.