A Resource for the Conditional Ablation of microRNAs in the Mouse

UCSF Diabetes Center, UCSF, San Francisco, CA 94143, USA.
Cell Reports (Impact Factor: 8.36). 04/2012; 1(4):385-91. DOI: 10.1016/j.celrep.2012.02.008
Source: PubMed

ABSTRACT The importance of miRNAs during development and disease processes is well established. However, most studies have been done in cells or with patient tissues, and therefore the physiological roles of miRNAs are not well understood. To unravel in vivo functions of miRNAs, we have generated conditional, reporter-tagged knockout-first mice for numerous evolutionarily conserved miRNAs. Here, we report the generation of 162 miRNA targeting vectors, 64 targeted ES cell lines, and 46 germline-transmitted miRNA knockout mice. In vivo lacZ reporter analysis in 18 lines revealed highly tissue-specific expression patterns and their miRNA expression profiling matched closely with published expression data. Most miRNA knockout mice tested were viable, supporting a mechanism by which miRNAs act redundantly with other miRNAs or other pathways. These data and collection of resources will be of value for the in vivo dissection of miRNA functions in mouse models.

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Available from: Jeffrey Bluestone, Dec 26, 2013
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    • "Procedures for the generation of miR- 200c/141 knockout mice were described previously (Park et al., 2012). Briefly, the miR-200c/141 knockout construct was generated by a previously described 'knockout-first' approach (Testa et al., 2004). "
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    ABSTRACT: The mouse incisor is a remarkable tooth that grows throughout the animal's lifetime. This continuous renewal is fueled by adult epithelial stem cells that give rise to ameloblasts, which generate enamel, and little is known about the function of microRNAs in this process. Here, we describe the role of a novel Pitx2:miR-200c/141:noggin regulatory pathway in dental epithelial cell differentiation. miR-200c repressed noggin, an antagonist of Bmp signaling. Pitx2 expression caused an upregulation of miR-200c and chromatin immunoprecipitation assays revealed endogenous Pitx2 binding to the miR-200c/141 promoter. A positive-feedback loop was discovered between miR-200c and Bmp signaling. miR-200c/141 induced expression of E-cadherin and the dental epithelial cell differentiation marker amelogenin. In addition, miR-203 expression was activated by endogenous Pitx2 and targeted the Bmp antagonist Bmper to further regulate Bmp signaling. miR-200c/141 knockout mice showed defects in enamel formation, with decreased E-cadherin and amelogenin expression and increased noggin expression. Our in vivo and in vitro studies reveal a multistep transcriptional program involving the Pitx2:miR-200c/141:noggin regulatory pathway that is important in epithelial cell differentiation and tooth development.
    Development 07/2013; 140(16). DOI:10.1242/dev.089193 · 6.27 Impact Factor
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    • "Other research centers have established mouse clinics, with the aim of carrying out a comprehensive analysis of the phenotypes of mutant lines of specific interest (e.g., Fuchs et al., 2012; Wakana et al., 2009; Laughlin et al., 2012). The genome-wide set of targeted mutations in ES cells established by the KOMP, EUCOMM, and MirKO programs (Skarnes et al., 2011; Prosser et al., 2011; Park et al., 2012) provides an opportunity to conduct systematic, large-scale gene function analysis in a mammalian system without the variables inherent in studies by individual groups. The Sanger Institute's Mouse Genetics Project (MGP) was one of the first programs to pursue this objective, established in 2006 when the first targeted ES cells became available. "
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    ABSTRACT: Mutations in whole organisms are powerful ways of interrogating gene function in a realistic context. We describe a program, the Sanger Institute Mouse Genetics Project, that provides a step toward the aim of knocking out all genes and screening each line for a broad range of traits. We found that hitherto unpublished genes were as likely to reveal phenotypes as known genes, suggesting that novel genes represent a rich resource for investigating the molecular basis of disease. We found many unexpected phenotypes detected only because we screened for them, emphasizing the value of screening all mutants for a wide range of traits. Haploinsufficiency and pleiotropy were both surprisingly common. Forty-two percent of genes were essential for viability, and these were less likely to have a paralog and more likely to contribute to a protein complex than other genes. Phenotypic data and more than 900 mutants are openly available for further analysis.
    07/2013; 154(2):452-464. DOI:10.1016/j.cell.2013.06.022
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    • "Procedures for the generation of miR- 200c/141 knockout mice were described previously (Park et al., 2012). Briefly, the miR-200c/141 knockout construct was generated by a previously described 'knockout-first' approach (Testa et al., 2004). "
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