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Berlin Brandenburg

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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: Proceedings of the National Academy of Sciences of the United States of America, 2012, Vol.109(24), pp.9354-9359
    Description: Gene targeting by zinc-finger nucleases in one-cell embryos provides an expedite mutagenesis approach in mice, rats, and rabbits. This technology has been recently used to create knockout and knockin mutants through the deletion or insertion of nucleotides. Here we apply zinc-finger nucleases in one-cell mouse embryos to generate disease-related mutants harboring single nucleotide or codon replacements. Using a gene-targeting vector or a synthetic oligodesoxynucleotide as template for homologous recombination, we introduced missense and silent mutations into the Rab38 gene, encoding a small GTPase that regulates intracellular vesicle trafficking. These results demonstrate the feasibility of seamless gene editing in one-cell embryos to create genetic disease models and establish synthetic oligodesoxynucleotides as a simplified mutagenesis tool. ; p. 9354-9359.
    Keywords: Nucleases ; Mice ; Physiological Transport ; Gene Targeting ; Rabbits ; Genes ; Disease Models ; Mutagenesis ; Genetic Disorders ; Nucleotides ; Homologous Recombination ; Guanosinetriphosphatase ; Rats ; Mutants
    ISSN: 0027-8424
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  • 3
    Language: English
    In: Proceedings of the National Academy of Sciences of the United States of America, 12 June 2012, Vol.109(24), pp.9354-9
    Description: Gene targeting by zinc-finger nucleases in one-cell embryos provides an expedite mutagenesis approach in mice, rats, and rabbits. This technology has been recently used to create knockout and knockin mutants through the deletion or insertion of nucleotides. Here we apply zinc-finger nucleases in one-cell mouse embryos to generate disease-related mutants harboring single nucleotide or codon replacements. Using a gene-targeting vector or a synthetic oligodesoxynucleotide as template for homologous recombination, we introduced missense and silent mutations into the Rab38 gene, encoding a small GTPase that regulates intracellular vesicle trafficking. These results demonstrate the feasibility of seamless gene editing in one-cell embryos to create genetic disease models and establish synthetic oligodesoxynucleotides as a simplified mutagenesis tool.
    Keywords: Embryo, Mammalian ; Gene Targeting ; Mutation
    ISSN: 00278424
    E-ISSN: 1091-6490
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  • 4
    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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  • 5
    Language: English
    In: Nucleic acids research, 27 July 2015, Vol.43(13), pp.6450-8
    Description: Using CRISPR/Cas9, it is possible to target virtually any gene in any organism. A major limitation to its application in gene therapy is the size of Cas9 (〉4 kb), impeding its efficient delivery via recombinant adeno-associated virus (rAAV). Therefore, we developed a split-Cas9 system, bypassing the packaging limit using split-inteins. Each Cas9 half was fused to the corresponding split-intein moiety and, only upon co-expression, the intein-mediated trans-splicing occurs and the full Cas9 protein is reconstituted. We demonstrated that the nuclease activity of our split-intein system is comparable to wild-type Cas9, shown by a genome-integrated surrogate reporter and by targeting three different endogenous genes. An analogously designed split-Cas9D10A nickase version showed similar activity as Cas9D10A. Moreover, we showed that the double nick strategy increased the homologous directed recombination (HDR). In addition, we explored the possibility of delivering the repair template accommodated on the same dual-plasmid system, by transient transfection, showing an efficient HDR. Most importantly, we revealed for the first time that intein-mediated split-Cas9 can be packaged, delivered and its nuclease activity reconstituted efficiently, in cells via rAAV.
    Keywords: Crispr-Cas Systems ; Inteins ; Crispr-Associated Proteins -- Genetics ; Deoxyribonucleases -- Genetics ; Genetic Therapy -- Methods
    ISSN: 03051048
    E-ISSN: 1362-4962
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  • 6
    In: genesis, June 2013, Vol.51(6), pp.448-455
    Description: Deregulated MAP kinase (MAPK) signaling plays key roles in developmental and adult disease processes, but the experimental activation of MAPK is a currently unresolved task. For the reversible induction of MAPK signaling, we generated transgenic mice harboring a tamoxifen inducible BRAFER fusion protein. The expression of the inducible BRAF kinase can be directed by Cre/loxP‐mediated recombination to selected cell types and enables the highly specific activation of MAPK signalling . We show that MAPK signaling can be transiently activated in the brain, liver, or kidney of BrafER mice by a single injection of tamoxifen. BrafER mice provide a new versatile tool to study disease mechanisms elicited by MAPK activation, complementing gene knockout technology that is restricted to the analysis of loss‐of‐function phenotypes. genesis 51:448–455. © 2013 Wiley Periodicals, Inc.
    Keywords: Mapk ; Braf ; Tamoxifen ; Estrogen Receptor ; Rosa26 ; Cre/Loxp ; Transgenic Mice
    ISSN: 1526-954X
    E-ISSN: 1526-968X
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  • 7
    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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  • 8
    In: Nature Protocols, 2013, Vol.8(12), p.2355
    Description: Genetically engineered mice are instrumental for the analysis of mammalian gene function in health and disease. As classical gene targeting, which is performed in embryonic stem (Es) cell cultures and generates chimeric mice, is a time- consuming and labor-intensive procedure, we recently used transcription activator-like (TAL) effector nucleases (TALENs) for mutagenesis of the mouse genome directly in one-cell embryos. Here we describe a stepwise protocol for the generation of knock-in and knockout mice, including the selection of TALEN-binding sites, the design and construction of TALEN coding regions and of mutagenic oligodeoxynucleotides (oDNs) and targeting vectors, mRNA production, embryo microinjection and the identification of modified alleles in founder mutants and their progeny. After a setup time of 2-3 weeks of hands-on work for TALEN construction, investigators can obtain first founder mutants for genes of choice within 7 weeks after embryo microinjections.
    Keywords: DNA Binding – Research ; Gene Mutation – Identification and Classification;
    ISSN: 1754-2189
    E-ISSN: 17502799
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  • 9
    Language: English
    In: FEBS Open Bio, 2015, Vol.5, pp.26-35
    Description: The use of TALEN and CRISPR/CAS nucleases is becoming increasingly popular as a means to edit single target sites in one-cell mouse embryos. Nevertheless, an area that has received less attention concerns the engineering of structural genome variants and the necessary religation of two distant double-strand breaks. Herein, we applied pairs of TALEN or sgRNAs and Cas9 to create deletions in the gene. We found that the deletion of 3.2 or 9.3 kb, but not of 30 kb, occurs at a frequency of 6–37%. This is sufficient for the direct production of mutants by embryo microinjection. Therefore, deletions up to ∼10 kb can be readily achieved for modeling human disease alleles. This work represents an important step towards the establishment of new protocols that support the ligation of remote DSB ends to achieve even larger rearrangements.
    Keywords: Talens ; Disease Model ; One-Cell Embryo ; Mouse Mutant ; Crispr ; Cas9 ; Biology
    ISSN: 2211-5463
    E-ISSN: 2211-5463
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  • 10
    Language: English
    In: Methods in molecular biology (Clifton, N.J.), 2016, Vol.1338, pp.229-43
    Description: Gene engineering for generating targeted mouse mutants is a key technology for biomedical research. Using TALENs as sequence-specific 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. By embryo microinjection of TALEN mRNAs and targeting vectors, knockout and knock-in alleles can be generated fast and efficiently. In this chapter we provide protocols for the application of TALENs in mouse zygotes.
    Keywords: Gene Targeting ; Knock-in ; Knockout ; Mouse ; One-Cell Embryo ; Talen ; Animals, Genetically Modified -- Genetics ; Endonucleases -- Genetics ; Gene Knockout Techniques -- Methods ; RNA Editing -- Genetics
    E-ISSN: 1940-6029
    Source: MEDLINE/PubMed (U.S. National Library of Medicine)
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