Journal
NATURE PROTOCOLS
Volume 8, Issue 12, Pages 2355-2379Publisher
NATURE PUBLISHING GROUP
DOI: 10.1038/nprot.2013.142
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Funding
- European Union within the EUCOMMTools project [HEALTH-F4-2010-261492]
- German Ministry of Education and Research within the DIGTOP project [01GS0858]
- German National Genome Research Network (NGFN)-Plus program
- Indian Council of Agricultural Research [29-1/2009-EQR/Edn]
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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 (ESES) 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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