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Wefers, B. ; Panda, S. ; Ortiz, O. ; Brandl, C. ; Hensler, S. ; Hansen, J. ; Wurst, W. ; Kühn, R.

Generation of targeted mouse mutants by embryo microinjection of TALEN mRNA.

Nat. Protoc. 8, 2355-2379 (2013)
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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.
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Publication type Article: Journal article
Document type Scientific Article
Keywords Zinc-finger Nucleases ; Homologous Recombination ; Effector Nucleases ; Mammalian-cells ; Iii Effectors ; Dna ; Genome ; Mice ; Mutations ; Frequency
Language english
Publication Year 2013
HGF-reported in Year 2013
ISSN (print) / ISBN 1754-2189
e-ISSN 1750-2799
Journal Nature Protocols
Quellenangaben Volume: 8, Issue: 12, Pages: 2355-2379 Article Number: , Supplement: ,
Publisher Nature Publishing Group
Reviewing status Peer reviewed
Institute(s) Institute of Developmental Genetics (IDG)
POF-Topic(s) 30204 - Cell Programming and Repair
Research field(s) Genetics and Epidemiology
PSP Element(s) G-500500-001
G-500500-003
DOI 10.1038/nprot.2013.142
WOS ID WOS:000328125800004
Scopus ID 85047687091
PubMed ID 24177293
Erfassungsdatum 2013-12-09
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