The generation of a conditional Fmr1 knock out mouse model to study Fmrp function in vivo

Article (PDF Available)inNeurobiology of Disease 21(3):549-55 · April 2006with45 Reads
DOI: 10.1016/j.nbd.2005.08.019 · Source: PubMed
Abstract
The FMR1 gene, mutated in Fragile X syndrome patients, has been modeled in mice with a neomycin cassette inserted in exon 5 of the mouse Fmr1 gene creating an Fmr1 knockout (Fmr1 KO) allele. This results in animals lacking Fmr1 protein (Fmrp) expression in all tissues. We have created a new, more versatile Fmr1 in vivo KO model (Fmr1 KO2) and generated conditional Fmr1 KO (CKO) mice by flanking the promoter and first exon of Fmr1 with lox P sites. This enables us to create a null allele in specific cell types and at specific time points by crossing Fmr1 CKO mice with tissue specific or inducible cre-recombinase expressing mice. The new Fmr1 KO2 line does not express any Fmrp and also lacks detectable Fmr1 transcripts. Crossing the Fmr1 CKO line with a Purkinje cell-specific cre-recombinase expresser produces mice that are null for Fmr1 in Purkinje neurons but wild type in all other cell types.

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Available from: Tao Zu, Nov 24, 2015
    • "Synaptosomal fractions were obtained from the hippocampi (n = 10/group) and cerebella (n = 10/group), and these samples were used for homogenization and stepwise centrifugation to remove cell debris and to extract the synaptosomes [13]. For neuronal cultures, E18 Fmr1 KO(2) and WT mouse embryos with a C57BL/6 background [11] were decapitated and both the hippocampi and the cerebella were dissected and used for neuronal cell culture (see Additional file 1, page 22). For electron microscopy, Purkinje cell-specific Fmr1 KO mice with a C57BL/6 background were generated via deletion of the first coding exon of Fmr1 through Cre-mediated recombination, as described by Koekkoek and colleagues [14]. "
    [Show abstract] [Hide abstract] ABSTRACT: Background: Fragile X syndrome (FXS) is a single-gene disorder that is the most common heritable cause of intellectual disability and the most frequent monogenic cause of autism spectrum disorders (ASD). FXS is caused by an expansion of trinucleotide repeats in the promoter region of the fragile X mental retardation gene (Fmr1). This leads to a lack of fragile X mental retardation protein (FMRP), which regulates translation of a wide range of messenger RNAs (mRNAs). The extent of expression level alterations of synaptic proteins affected by FMRP loss and their consequences on synaptic dynamics in FXS has not been fully investigated. Methods: Here, we used an Fmr1 knockout (KO) mouse model to investigate the molecular mechanisms underlying FXS by monitoring protein expression changes using shotgun label-free liquid-chromatography mass spectrometry (LC-MSE) in brain tissue and synaptosome fractions. FXS-associated candidate proteins were validated using selected reaction monitoring (SRM) in synaptosome fractions for targeted protein quantification. Furthermore, functional alterations in synaptic release and dynamics were evaluated using live-cell imaging, and interpretation of synaptic dynamics differences was investigated using electron microscopy. Results: Key findings relate to altered levels of proteins involved in GABA-signalling, especially in the cerebellum. Further exploration using microscopy studies found reduced synaptic vesicle unloading of hippocampal neurons and increased vesicle unloading in cerebellar neurons, which suggests a general decrease of synaptic transmission. Conclusions: Our findings suggest that FMRP is a regulator of synaptic vesicle dynamics, which supports the role of FMRP in presynaptic functions. Taken together, these studies provide novel insights into the molecular changes associated with FXS.
    Full-text · Article · Dec 2016
    • "To specifically investigate how astroglial FMRP may modulate neuronal deficits in FXS in vivo, we generated inducible astrocytespecific Fmr1 cKO (deletion) and cON (restoration) mice by breeding Fmr1 f/f (and Fmr1 f/ ) or Fmr1 loxP-neo/loxP-neo with BAC GLAST CreERT transgenic mice (Fig. 1B, C). The Fmr1 f/y mice show no difference in phenotype compared with WT mice, while Fmr1 loxP-neo/y mice express very low levels (810%) of FMRP and show phenotypes that are highly similar to those of Fmr1 KO (Fmr1 /y ) mice (Mientjes et al., 2006 ). FMRP levels can be restored in a Cre-dependent manner following deletion of the inserted neo gene from the fmr1 allele in Fmr1 loxP-neo/y mice. "
    [Show abstract] [Hide abstract] ABSTRACT: Significance statement: Previous studies to understand how the loss of function of fragile X mental retardation protein (FMRP) causes fragile X syndrome (FXS) have largely focused on neurons; whether the selective loss of astroglial FMRP in vivo alters astrocyte functions and contributes to the pathogenesis of FXS remain essentially unknown. This has become a long-standing unanswered question in the fragile X field, which is also relevant to autism pathogenesis. Our current study generated astrocyte-specific Fmr1 conditional knock-out and restoration mice, and provided compelling evidence that the selective loss of astroglial FMRP contributes to cortical synaptic deficits in FXS, likely through the dysregulated astroglial glutamate transporter GLT1 expression and impaired glutamate uptake. These results demonstrate previously undescribed astrocyte-mediated mechanisms in the pathogenesis of FXS.
    Full-text · Article · Jul 2016
    • "Also, a second vector was used that targeted exons 7–9, which encode parts of the EGF domain (carboxy-terminus) of NGR1, was replaced by a neomycin resistance gene (Meyer and Birchmeier, 1995). The matings between heterozygous mice carrying either mutation produced no homozygous mutant offspring, indicating that the complete deletion of NRG1 and its receptor, ErbB, are lethal during embryogenesis due to heart malformations and defects in Schwann cells and cranial glia development (Meyer and Birchmeier, 1995). Therefore, NRG1 and its ErbB receptor are indispensable during development. "
    [Show abstract] [Hide abstract] ABSTRACT: Over the past three decades, genetic manipulations in mice have been used in neuroscience as a major approach to investigate the in vivo function of genes and their alterations. In particular, gene targeting techniques using embryonic stem cells have revolutionized the field of mammalian genetics and have been at the forefront in the generation of numerous mouse models of human brain disorders. In this review, we will first examine childhood developmental disorders such as autism, intellectual disability, Fragile X syndrome, and Williams-Beuren syndrome. We will then explore psychiatric disorders such as schizophrenia and lastly, neurodegenerative disorders including Alzheimer's disease and Parkinson's disease. We will outline the creation of these mouse models that range from single gene deletions, subtle point mutations to multi-gene manipulations, and discuss the key behavioral phenotypes of these mice. Ultimately, the analysis of the models outlined in this review will enhance our understanding of the in vivo role and underlying mechanisms of disease-related genes in both normal brain function and brain disorders, and provide potential therapeutic targets and strategies to prevent and treat these diseases.
    Full-text · Article · Mar 2016
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