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Open Access Green as soon as Postprint is submitted to ZB.
Generation of targeted mouse mutants by embryo microinjection of TALEN mRNA.
Nat. Protoc. 8, 2355-2379 (2013)
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
ISSN (print) / ISBN
1754-2189
e-ISSN
1750-2799
Journal
Nature Protocols
Quellenangaben
Volume: 8,
Issue: 12,
Pages: 2355-2379
Publisher
Nature Publishing Group
Non-patent literature
Publications
Reviewing status
Peer reviewed
Institute(s)
Institute of Developmental Genetics (IDG)