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  • 1
    Language: English
    In: Proceedings of the National Academy of Sciences of the United States of America, 05 March 2013, Vol.110(10), pp.3782-7
    Description: The study of genetic disease mechanisms relies mostly on targeted mouse mutants that are derived from engineered embryonic stem (ES) cells. Nevertheless, the establishment of mutant ES cells is laborious and time-consuming, restricting the study of the increasing number of human disease mutations discovered by high-throughput genomic analysis. Here, we present an advanced approach for the production of mouse disease models by microinjection of transcription activator-like effector nucleases (TALENs) and synthetic oligodeoxynucleotides into one-cell embryos. Within 2 d of embryo injection, we created and corrected chocolate missense mutations in the small GTPase RAB38; a regulator of intracellular vesicle trafficking and phenotypic model of Hermansky-Pudlak syndrome. Because ES cell cultures and targeting vectors are not required, this technology enables instant germline modifications, making heterozygous mutants available within 18 wk. The key features of direct mutagenesis by TALENs and oligodeoxynucleotides, minimal effort and high speed, catalyze the generation of future in vivo models for the study of human disease mechanisms and interventions.
    Keywords: Disease Models, Animal ; Germ-Line Mutation ; Genetic Diseases, Inborn -- Genetics ; Oligodeoxyribonucleotides -- Administration & Dosage
    ISSN: 00278424
    E-ISSN: 1091-6490
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  • 2
    Language: English
    In: Genetics, November 2013, Vol.195(3), pp.703-13
    Description: Targeted mouse mutants are instrumental for the analysis of gene function in health and disease. We recently provided proof-of-principle for the fast-track mutagenesis of the mouse genome, using transcription activator-like effector nucleases (TALENs) in one-cell embryos. Here we report a routine procedure for the efficient production of disease-related knockin and knockout mutants, using improved TALEN mRNAs that include a plasmid-coded poly(A) tail (TALEN-95A), circumventing the problematic in vitro polyadenylation step. To knock out the C9orf72 gene as a model of frontotemporal lobar degeneration, TALEN-95A mutagenesis induced sequence deletions in 41% of pups derived from microinjected embryos. Using TALENs together with mutagenic oligodeoxynucleotides, we introduced amyotrophic lateral sclerosis patient-derived missense mutations in the fused in sarcoma (Fus) gene at a rate of 6.8%. For the simple identification of TALEN-induced mutants and their progeny we validate high-resolution melt analysis (HRMA) of PCR products as a sensitive and universal genotyping tool. Furthermore, HRMA of off-target sites in mutant founder mice revealed no evidence for undesired TALEN-mediated processing of related genomic sequences. The combination of TALEN-95A mRNAs for enhanced mutagenesis and of HRMA for simplified genotyping enables the accelerated, routine production of new mouse models for the study of genetic disease mechanisms.
    Keywords: C9orf72 ; Fus ; Talens ; Disease Model ; Mouse Mutant ; Nuclease ; One-Cell Embryo ; Mutagenesis ; Genetic Engineering -- Methods
    ISSN: 00166731
    E-ISSN: 1943-2631
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  • 3
    In: Nature Biotechnology, 2015
    Description: The insertion of precise genetic modifications by genome editing tools such as CRISPR-Cas9 is limited by the relatively low efficiency of homology-directed repair (HDR) compared with the higher efficiency of the nonhomologous end-joining (NHEJ) pathway. To enhance HDR, enabling the insertion of precise genetic modifications, we suppressed the NHEJ key molecules KU70, KU80 or DNA ligase IV by gene silencing, the ligase IV inhibitor SCR7 or the coexpression of adenovirus 4 E1B55K and E4orf6 proteins in a 'traffic light' and other reporter systems. Suppression of KU70 and DNA ligase IV promotes the efficiency of HDR 4-5-fold. When co-expressed with the Cas9 system, E1B55K and E4orf6 improved the efficiency of HDR up to eightfold and essentially abolished NHEJ activity in both human and mouse cell lines. Our findings provide useful tools to improve the frequency of precise gene modifications in mammalian cells.
    Keywords: Crispr-Cas Systems -- Genetics ; DNA End-Joining Repair -- Genetics ; Genetic Engineering -- Methods;
    ISSN: 1087-0156
    E-ISSN: 15461696
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  • 4
    Language: English
    In: Methods, 15 August 2014, Vol.69(1), pp.94-101
    Description: Gene engineering for generating targeted mouse mutants is a key technology for biomedical research. Using TALENs as nucleases to induce targeted double-strand breaks, the mouse genome can be directly modified in zygotes in a single step, without the need for embryonic stem cells. Thereby, knockout and knockin alleles can be generated fast and efficiently by embryo microinjection of TALEN mRNAs and targeting vectors. In this article we present an introduction into the TALEN technology and provide protocols for the application of TALENs in mouse zygotes.
    Keywords: Knockin ; Knockout ; One-Cell Embryo ; Gene Targeting ; Talen ; Mouse ; Chemistry ; Anatomy & Physiology
    ISSN: 1046-2023
    E-ISSN: 1095-9130
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  • 5
    Language: English
    In: Nature biotechnology, 06 February 2018, Vol.36(2), pp.196
    Keywords: Genetic Modification ; Crispr ; Homology ; Mammalian Cells ; Homology;
    ISSN: 10870156
    E-ISSN: 1546-1696
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  • 6
    Language: English
    In: Methods, 15 May 2017, Vol.121-122, pp.55-67
    Description: The generation of targeted mouse mutants is a key technology for biomedical research. Using the CRISPR/Cas9 system for induction of targeted double-strand breaks, gene editing can be performed in a single step directly in mouse zygotes. This article covers the design of knockout and knockin alleles, preparation of reagents, microinjection or electroporation of zygotes and the genotyping of pups derived from gene editing projects. In addition we include a section for the control of experimental settings by targeting the Rosa26 locus and PCR based genotyping of blastocysts.
    Keywords: Crispr ; Cas9 ; Mouse ; Zygotes ; Gene Editing ; Chemistry ; Anatomy & Physiology
    ISSN: 1046-2023
    E-ISSN: 1095-9130
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  • 7
    Language: English
    In: PLoS ONE, 2012, Vol.7(4), p.e35035
    Description: MAP kinase signaling has been implicated in brain development, long-term memory, and the response to antidepressants. Inducible Braf knockout mice, which exhibit protein depletion in principle forebrain neurons, enabled us to unravel a new role of neuronal MAPK signaling for emotional behavior. Braf mice that were induced during adulthood showed normal anxiety but increased depression-like behavior, in accordance with pharmacological findings. In contrast, the inducible or constitutive inactivation of Braf in the juvenile brain leads to normal depression-like behavior but decreased anxiety in adults. In juvenile, constitutive mutants we found no alteration of GABAergic neurotransmission but reduced neuronal arborization in the dentate gyrus. Analysis of gene expression in the hippocampus revealed nine downregulated MAPK target genes that represent candidates to cause the mutant phenotype. ; Our results reveal the differential function of MAPK signaling in juvenile and adult life phases and emphasize the early postnatal period as critical for the determination of anxiety in adults. Moreover, these results validate inducible gene inactivation as a new valuable approach, allowing it to discriminate between gene function in the adult and the developing postnatal brain.
    Keywords: Research Article ; Biology ; Medicine ; Social And Behavioral Sciences ; Genetics And Genomics ; Molecular Biology ; Mental Health ; Physiology ; Neuroscience
    E-ISSN: 1932-6203
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  • 8
    Language: English
    In: Methods, 15 August 2014, Vol.69(1), pp.1-1
    Keywords: Chemistry ; Anatomy & Physiology
    ISSN: 1046-2023
    E-ISSN: 1095-9130
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  • 9
    Language: English
    In: Current protocols in mouse biology, 01 March 2011, Vol.1(1), pp.199-211
    Description: This unit provides an overview of the major types of mutant alleles that can be generated by gene targeting in ES cells. It presents the growing public resources of premade gene targeting vectors, modified ES cells, and mutant mice. General guidelines for the design of targeting vectors are followed by protocols for the construction of vectors to generate knockout (KO), conditional KO, and subtle mutant alleles. Curr. Protoc. Mouse Biol. 1:199-211. © 2011 by John Wiley & Sons, Inc.
    Keywords: Eucomm/Komp ; Conditional Ko Mice ; Gene Targeting ; Knockout Mice ; Targeting Vector Design
    ISSN: 2161-2617
    Source: MEDLINE/PubMed (U.S. National Library of Medicine)
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  • 10
    Book
    Book
    Springer New York, New York, NY
    Language: English
    Keywords: Biomedicine -- Human Genetics; Biomedicine -- Animal Genetics and Genomics; Biomedicine -- Animal Models
    ISBN: 978-1-4939-2931-3
    Source: Springer Science & Business Media B.V.
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