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Berlin Brandenburg

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  • Meyer, Melanie  (6)
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  • 1
    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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  • 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, 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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  • 4
    Language: English
    In: Proceedings of the National Academy of Sciences of the United States of America, 24 August 2010, Vol.107(34), pp.15022-6
    Description: Gene targeting by homologous recombination in embryonic stem cells is extensively used to generate specific mouse mutants. However, most mammalian species lack tools for targeted gene manipulation. Since double-strand breaks strongly increase the rate of homologous recombination at genomic loci, we explored whether gene targeting can be directly performed in zygotes by the use of zinc-finger nucleases. Here we report that gene targeting is achieved in 1.7-4.5% of murine one-cell embryos upon the coinjection of targeting vectors with zinc-finger nucleases, without preselection. These findings enable the manipulation of the mammalian germ line in a single step in zygotes, independent of ES cells.
    Keywords: Endonucleases -- Metabolism ; Gene Targeting -- Methods ; Zygote -- Metabolism
    ISSN: 00278424
    E-ISSN: 1091-6490
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  • 5
    Language: German
    In: BIOspektrum, 2011, Vol.17(5), pp.537-540
    Description: In vielen Säugetierarten gab es bis jetzt keine effektiven Werkzeuge für eine gezielte Genmodifikation. Die Entwicklung der Zinkfingernuklease-Technologie ermöglicht nun auch in diesen Organismen eine schnelle und einfache Genommanipulation. Most mammalian species lack useful tools for targeted gene modification. With the discovery of the zinc-finger nuclease technology it is now possible to achieve rapid and easy genome manipulations in mammals.
    Keywords: Life Sciences ; Biochemistry, General ; Life Sciences, General ; Human Genetics ; Developmental Biology ; Pharmacology/Toxicology ; Microbiology ; Sciences (General);
    ISSN: 0947-0867
    E-ISSN: 1868-6249
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  • 6
    Language: English
    In: Current protocols in mouse biology, 01 December 2012, Vol.2(4), pp.347-64
    Description: Gene targeting by sequence-specific nucleases in one-cell embryos provides an expedited mutagenesis approach in rodents. This technology has been recently established to create knockout and knockin mutants through sequence deletion or sequence insertion. This article provides protocols for the preparation and microinjection of nuclease mRNA and targeting vector DNA into fertilized mouse eggs. Furthermore, we provide guidelines for genotyping the desired mouse mutants. Curr. Protoc. Mouse Biol. 2:347-364 © 2012 by John Wiley & Sons, Inc.
    Keywords: Tal Nuclease ; Gene Targeting ; Homologous Recombination ; Mouse Mutant ; Pronucleus Injection ; Zinc-Finger Nuclease
    ISSN: 2161-2617
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