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Generation of targeted mouse mutants by embryo microinjection of TALEN mRNA

Item Type:Article
Title:Generation of targeted mouse mutants by embryo microinjection of TALEN mRNA
Creators Name:Wefers, B. and Panda, S.K. and Ortiz, O. and Brandl, C. and Hensler, S. and Hansen, J. and Wurst, W. and Kühn, R.
Abstract: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:Binding Sites, Deoxyribonucleases, Gene Targeting, Genetic Engineering, Genotyping Techniques, Mammalian Embryo, Messenger RNA, Microinjections, Oligodeoxyribonucleotides, Site-Directed Mutagenesis, Animals, Mice
Source:Nature Protocols
Publisher:Nature Publishing Group
Page Range:2355-2379
Date:December 2013
Official Publication:https://doi.org/10.1038/nprot.2013.142
PubMed:View item in PubMed

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