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


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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    • "Both morphants showed a reduction in hair cell number and different abnormalities in inner ear structure, indicating that the two miRNAs act in parallel but different pathways. Additional models may be obtained from a resource of miRNA reporter and conditional knockout mouse lines (Park et al., 2012). "
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    ABSTRACT: Non-coding microRNAs (miRNAs) have a fundamental role in gene regulation and expression in almost every multicellular organism. Only discovered in the last decade, miRNAs are already known to play a leading role in many aspects of disease. In the vertebrate inner ear, miRNAs are essential for controlling development and survival of hair cells. Moreover, dysregulation of miRNAs has been implicated in sensorineural hearing impairment, as well as in other ear diseases such as cholesteatomas, vestibular schwannomas, and otitis media. Due to the inaccessibility of the ear in humans, animal models have provided the optimal tools to study miRNA expression and function, in particular mice and zebrafish. A major focus of current research has been to discover the targets of the miRNAs expressed in the inner ear, in order to determine the regulatory pathways of the auditory and vestibular systems. The potential for miRNAs manipulation in development of therapeutic tools for hearing impairment is as yet unexplored, paving the way for future work in the field.
    Frontiers in Molecular Neuroscience 12/2013; 6:52. DOI:10.3389/fnmol.2013.00052 · 4.08 Impact Factor
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    • "This study describes a single miRNA knockout that exhibits severe postnatal defects that appear within two weeks of birth. Most published miRNA knockouts lack such severe phenotypes, and those with a lethality often die embryonically or soon after birth [21]. These data support a role for miR-205 in squamous epithelium derived organs, suggested by a bevy of work aimed at delineating the targets and pathways affected by miR-205 activity. "
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    ABSTRACT: MicroRNAs are small noncoding RNAs thought to have pivotal roles in numerous diseases and developmental processes. However, a growing body of literature indicates that in vivo elimination of these tiny RNAs usually has little to no observable consequence, suggesting functional redundancy with other microRNAs or cellular pathways. We provide an in-depth analysis of miR-205 expression and define miR-205 as an epithelial-specific microRNA, and for the first time show that ablation of this microRNA knockout exhibits partially penetrant lethality in a constitutive mouse knockout model. Given the role of this microRNA in cancer and development, this mouse model will be an incredible reagent to study the function and mechanisms of miR-205 in epithelial tissue development and disease.
    PLoS ONE 10/2013; 8(10):e76634. DOI:10.1371/journal.pone.0076634 · 3.23 Impact Factor
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    • "The miRNA OpenArray® screen also identified high expression of miR-376a in chondrocytes throughout the human developing limb. This miRNA is part of a large cluster, two of which (miR-654/376b) were found to be expressed strongly in murine embryonic cartilage [74]. Our studies also showed that miR-127 and miR-409-3p were strongly expressed at all stages of human embryonic chondrocyte differentiation. "
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    ABSTRACT: There is compelling in vivo evidence from reports on human genetic mutations and transgenic mice that some microRNAs (miRNAs) play an important functional role in regulating skeletal development and growth. A number of published in vitro studies also point toward a role for miRNAs in controlling chondrocyte gene expression and differentiation. However, information on miRNAs that may regulate a specific phase of chondrocyte differentiation (i.e. production of progenitor, differentiated or hypertrophic chondrocytes) is lacking. To attempt to bridge this knowledge gap, we have investigated miRNA expression patterns in human embryonic cartilage tissue. Specifically, a developmental time point was selected, prior to endochondral ossification in the embryonic limb, to permit analysis of three distinct populations of chondrocytes. The location of chondroprogenitor cells, differentiated chondrocytes and hypertrophic chondrocytes in gestational day 54-56 human embryonic limb tissue sections was confirmed both histologically and by specific collagen expression patterns. Laser capture microdissection was utilized to separate the three chondrocyte populations and a miRNA profiling study was carried out using TaqMan® OpenArray® Human MicroRNA Panels (Applied Biosystems®). Here we report on abundantly expressed miRNAs in human embryonic cartilage tissue and, more importantly, we have identified miRNAs that are significantly differentially expressed between precursor, differentiated and hypertrophic chondrocytes by 2-fold or more. Some of the miRNAs identified in this study have been described in other aspects of cartilage or bone biology, while others have not yet been reported in chondrocytes. Finally, a bioinformatics approach was applied to begin to decipher developmental cellular pathways that may be regulated by groups of differentially expressed miRNAs during distinct stages of chondrogenesis. Data obtained from this work will serve as an important resource of information for the field of cartilage biology and will enhance our understanding of miRNA-driven mechanisms regulating cartilage and endochondral bone development, regeneration and repair.
    PLoS ONE 09/2013; 8(9):e75012. DOI:10.1371/journal.pone.0075012 · 3.23 Impact Factor
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