title: The use of TALENs for nonhomologous end joining mutagenesis in silkworm and fruitfly.
abstract: Transcription activator-like effector nucleases (TALENs) are custom-made enzymes designed to cut double-stranded DNA at desired locations. The DNA breaks are repaired either by error-prone non-homologous end-joining (NHEJ) pathway or via homologous recombination requiring homologous DNA as a template for the repair. TALENs are used for site-specific mutagenesis in an extended range of organisms including insects. We will describe here a simple TALEN-based mutagenesis protocol suitable for the generation of germline mutations in Bombyx mori and Drosophila melanogaster. The protocol includes assembly of specific TAL modules, in vitro synthesis of TALEN RNAs, egg microinjection and mutation detection using PCR analysis. Our procedure allows a high frequency induction of NHEJ mutations, which often allows the reception of homozygous mutants already in the G1.
authors.affiliation: National Institute of Agrobiological Sciences, 1-2 Owashi, Tsukuba, Ibaraki 305-8634, Japan.
authors.forename: Yoko
authors.initials: Y
authors.lastname: Takasu
authors.affiliation: National Institute of Agrobiological Sciences, 1-2 Owashi, Tsukuba, Ibaraki 305-8634, Japan.
authors.forename: Toshiki
authors.initials: T
authors.lastname: Tamura
authors.affiliation: Biology Centre of the ASCR, Branisovska 31, 370 05 Ceske Budejovice, Czech Republic.
authors.forename: Suresh
authors.initials: S
authors.lastname: Sajwan
authors.affiliation: National Institute of Agrobiological Sciences, 1-2 Owashi, Tsukuba, Ibaraki 305-8634, Japan.
authors.forename: Isao
authors.initials: I
authors.lastname: Kobayashi
authors.affiliation: Biology Centre of the ASCR, Branisovska 31, 370 05 Ceske Budejovice, Czech Republic. Electronic address: zurovec@entu.cas.cz.
authors.forename: Michal
authors.initials: M
authors.lastname: Zurovec
mesh_terms: D000818:Animals; D001483:Base Sequence; D012831:Bombyx / Q000235:genetics*; D053903:DNA Breaks, Double-Stranded; D059766:DNA End-Joining Repair; D003851:Deoxyribonucleases / Q000235:genetics; D004331:Drosophila melanogaster / Q000235:genetics*; D008845:Microinjections / Q000295:instrumentation; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed / Q000379:methods*
chemical_list: D003851:Deoxyribonucleases
keywords: Bombyx mori; Drosophila melanogaster; Engineered nucleases; Genotyping; Golden Gate assembly; pBlue-TAL