title: Generation of targeted mouse mutants by embryo microinjection of TALEN mRNA.
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.
authors.affiliation: Institute of Developmental Genetics, Helmholtz Zentrum München, German Research Center for Environmental Health, Munich, Germany.
authors.forename: Benedikt
authors.initials: B
authors.lastname: Wefers
authors.forename: Sudeepta K
authors.initials: SK
authors.lastname: Panda
authors.forename: Oskar
authors.initials: O
authors.lastname: Ortiz
authors.forename: Christina
authors.initials: C
authors.lastname: Brandl
authors.forename: Svenja
authors.initials: S
authors.lastname: Hensler
authors.forename: Jens
authors.initials: J
authors.lastname: Hansen
authors.forename: Wolfgang
authors.initials: W
authors.lastname: Wurst
authors.forename: Ralf
authors.initials: R
authors.lastname: Kühn
mesh_terms: D000818:Animals; D001665:Binding Sites; D003851:Deoxyribonucleases / Q000737:chemistry / Q000235:genetics; D004622:Embryo, Mammalian; D018390:Gene Targeting / Q000379:methods*; D005818:Genetic Engineering / Q000379:methods; D060005:Genotyping Techniques; D051379:Mice; D018345:Mice, Knockout / Q000235:genetics*; D008845:Microinjections / Q000379:methods; D016297:Mutagenesis, Site-Directed / Q000379:methods*; D009838:Oligodeoxyribonucleotides / Q000138:chemical synthesis; D012333:RNA, Messenger / Q000737:chemistry*
chemical_list: D009838:Oligodeoxyribonucleotides; D012333:RNA, Messenger; D003851:Deoxyribonucleases