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The Odd-skipped related 1 (Osr1) gene encodes a zinc finger protein homologous to the Drosophila Odd-skipped transcription factor. During mouse embryogenesis, Osr1 is expressed in multiple tissues, including the developing heart, kidney, limb, lung, and craniofacial structures. While characterization of targeted mutant mice has revealed essential roles for Osr1 in heart and kidney development, the embryonic lethality of the Osr1 null mice has hindered investigation of its role in many other developmental processes. We report here the generation of conditional mutant mice containing two loxP sites flanking Exon 2 of the Osr1 gene. Mice homozygous for this targeted Osr1fneo allele are normal and fertile. Crossing the Osr1fneo/fneo mice with the EIIa-Cre transgenic mice resulted in Cre-mediated deletion of the loxP-flanked Exon2 in the germ line and mice homozygous for this deletion recapitulated the Osr1 null mutant phenotypes. The Osr1fneo conditional mice will be valuable for tissue-specific analysis of the roles of Osr1 in embryonic and postnatal developmental processes.
The Odd-skipped family of zinc finger transcription factors plays essential roles in embryonic development throughout metazoans. The odd-skipped gene was first identified in a large mutagenesis screen of developmental control genes in Drosophila as mutations in this gene caused loss of portions of the odd-numbered segments in the embryo (Nusslein-Volhard and Wieschaus, 1980; Coulter and Wieschaus, 1988). Since the characterization of the coding sequences of the odd-skipped gene, homologous genes have been identified in mice, human, chick, nematode, Xenopus, and zebrafish (Coulter et al., 1990; So and Danielian, 1999; Lan et al., 2001; Katoh, 2002; Buckley et al., 2004; Stricker et al., 2006; Tena et al., 2007; Mudumana et al., 2008). Analysis of gene knockout mice showed that Odd-skipped related-1 (Osr1) is essential for heart and kidney development whereas Osr2 plays critical roles in palate and tooth development (Lan et al., 2004; Wang et al., 2005; James et al., 2006; Mugford et al., 2008; Zhang et al., 2009). Gene knockdown studies in Xenopus and zebrafish showed that both Osr1 and Osr2 play critical roles in embryonic kidney development in these organisms (Tena et al., 2007; Mudumana et al., 2008).
During mouse embryogenesis, Osr1 mRNA expression is first activated in the nascent intermediate mesoderm during gastrulation and subsequently in the developing heart, limb, lung, and craniofacial structures (Wang et al., 2005). While analyses of the Osr1 null mutant mouse strains have revealed essential roles for Osr1 in the early stages of kidney and heart development, the embryonic lethality of the mutant mice has hindered investigations of the roles of Osr1 in many other developmental processes (Wang et al., 2005). To overcome this problem, we have generated mice for conditional inactivation of the Osr1 gene using the Cre-loxP in vivo DNA recombination system.
The mouse Osr1 gene contains three exons, spanning approximately 8 kb of genomic DNA sequence (FIG. 1A) (Wang et al., 2005). We isolated an Osr1 genomic DNA fragment containing all three exons from the RPCI-22 129/SvEvTac mouse BAC library (BACPAC Resources, Children’s Hospital of Oakland, Oakland, CA). To construct the targeting vector for generation of Osr1 conditional mice, one loxP sequence was inserted into each of the two EcoRV sites in the first and second introns, respectively (FIG. 1B). An frt-flanked neo expression cassette was inserted immediately 3′ to the loxP sequence in Intron 1 for positive selection. The resultant targeting vector contained the 3.8 kb BglII-EcoRI fragment from Intron 1 as the 5′ homology arm, the 3.2 kb EcoRV-XbaI fragment containing Exon 3 as the 3′ homology arm, and a diphtheria toxin-A (DTA) expression cassette immediately 3′ to the 3′ arm (FIG. 1B). Following sequence confirmation of the construct, the targeting vector was linearized and electroporated into the CJ7 mouse embryonic stem (ES) cells as previously described (Swiatek and Gridley, 1997). G418 resistant ES colonies were screened by Southern hybridization with the 3′ probe to detect integration of the 3′ loxP site in the second intron (FIG. 1F). Southern blots containing DNA samples of positively targeted clones were rehybridized with the 5′ probe (indicated in FIG. 1A) to verify the expected integration of the loxP and neo sequences in the first intron (data not shown). Of 224 colonies screened, three correctly targeted ES clones were identified and two independent clones were used for generation of chimeric mice by microinjection into blastocysts of C57BL/6J mice. Chimeric male mice were bred with C57BL/6J females to test for germ line transmission of the targeted allele. Both ES clones resulted in germ line transmission. F1 mice were genotyped initially by Southern hybridization analysis using tail genomic DNA samples for the presence of the targeted Osr1fneo allele (FIG. 1C). Heterozygous mice were intercrossed and genotyping of F2 progeny at one week after birth detected homozygous Osr1fneo/fneo mice at expected Mendelian ratio. The homozygous Osr1fneo/fneo mice exhibited normal life span and fertility. Some homozygous Osr1fneo/fneo mice were bred to FLPeR mice (Farley et al., 2000) to remove the neo expression cassette in the germ line (FIG. 1D). The resultant Osr1f/+ mice were intercrossed and homozygous Osr1f/f mice were generated and did not exhibit any detectable abnormality.
To test for Cre mediated inactivation of the Osr1 conditional allele in vivo, we crossed the Osr1fneo/fneo mice with EIIa-Cre transgenic mice, which express Cre in the early embryo and causes deletion of loxP-flanked sequences in most tissues including the germ line (Lakso et al., 1996). Whereas the resultant Osr1ΔE2/+ mice were normal, analysis of embryos from Osr1ΔE2/+ heterozygous intercrosses revealed that the Osr1ΔE2/ΔE2 mutant embryos died before birth (FIG. 2, FIG. 3). The Osr1ΔE2/ΔE2 embryos phenocopied the phenotypes of the Osr1−/− mutant embryos as previously reported (Wang et al., 2005), with most of them dying at around E12.0 (FIG. 3, B and D). Similar to the Osr1−/− embryos, a small percentage of Osr1ΔE2/ΔE2 embryos survived to late gestation and had generalized edema (FIG. 3E). Histological analysis showed that all Osr1ΔE2/ΔE2 embryos lacked metanephric kidney and had heart defects (data not shown), similar to the Osr1−/− embryos as previously reported (Wang et al., 2005). These data indicate that Cre-mediated deletion of the loxP-flanked Exon-2 abolished Osr1 gene function. Thus, the Osr1fneo conditional mice will be valuable for tissue-specific genetic analysis of the molecular pathways involving Osr1 in many developmental processes.
We thank the University of Rochester Transgenic Mouse Facility for generating chimeric founder mice from our gene-targeted mouse embryonic stem cells. This work was supported by NIH/NIDCR grant R01DE013681.