title: Targeted gene disruption by use of transcription activator-like effector nuclease (TALEN) in the water flea Daphnia pulex.
abstract: BACKGROUND
  The cosmopolitan microcrustacean Daphnia pulex provides a model system for both human health research and monitoring ecosystem integrity. It is the first crustacean to have its complete genome sequenced, an unprecedented ca. 36% of which has no known homologs with any other species. Moreover, D. pulex is ideally suited for experimental manipulation because of its short reproductive cycle, large numbers of offspring, synchronization of oocyte maturation, and other life history characteristics. However, existing gene manipulation techniques are insufficient to accurately define gene functions. Although our previous investigations developed an RNA interference (RNAi) system in D. pulex, the possible time period of functional analysis was limited because the effectiveness of RNAi is transient. Thus, in this study, we developed a genome editing system for D. pulex by first microinjecting transcription activator-like effector nuclease (TALEN) mRNAs into early embryos and then evaluating TALEN activity and mutation phenotypes.
  
  
  RESULTS
  We assembled a TALEN construct specific to the Distal-less gene (Dll), which is a homeobox transcription factor essential for distal limb development in invertebrates and vertebrates, and evaluated its activity in vitro by single-strand annealing assay. Then, we injected TALEN mRNAs into eggs within 1 hour post-ovulation. Injected embryos presented with defects in the second antenna and altered appendage development, and indel mutations were detected in Dll loci, indicating that this technique successfully knocked out the target gene.
  
  
  CONCLUSIONS
  We succeeded, for the first time in D. pulex, in targeted mutagenesis by use of Platinum TALENs. This genome editing technique makes it possible to conduct reverse genetic analysis in D. pulex, making this species an even more appropriate model organism for environmental, evolutionary, and developmental genomics.
authors.affiliation: Okazaki Institute for Integrative Bioscience, National Institute for Basic Biology, National Institutes of Natural Sciences, Higashiyama 5-1, Myodaiji, Okazaki, Aichi, 444-8787, Japan. chizueh@nibb.ac.jp.
authors.forename: Chizue
authors.initials: C
authors.lastname: Hiruta
authors.affiliation: Okazaki Institute for Integrative Bioscience, National Institute for Basic Biology, National Institutes of Natural Sciences, Higashiyama 5-1, Myodaiji, Okazaki, Aichi, 444-8787, Japan. ogino@nibb.ac.jp.|Faculty of Life Science, Graduate University for Advanced Studies (SOKENDAI), 5-1 Higashiyama, Myodaiji, Okazaki, Aichi, 444-8787, Japan. ogino@nibb.ac.jp.
authors.forename: Yukiko
authors.initials: Y
authors.lastname: Ogino
authors.affiliation: Department of Mathematical and Life Sciences, Graduate School of Science, Hiroshima University, Higashi-Hiroshima, 739-8526, Japan. tetsushi-sakuma@hiroshima-u.ac.jp.
authors.forename: Tetsushi
authors.initials: T
authors.lastname: Sakuma
authors.affiliation: Okazaki Institute for Integrative Bioscience, National Institute for Basic Biology, National Institutes of Natural Sciences, Higashiyama 5-1, Myodaiji, Okazaki, Aichi, 444-8787, Japan. kenji514@nibb.ac.jp.|Faculty of Life Science, Graduate University for Advanced Studies (SOKENDAI), 5-1 Higashiyama, Myodaiji, Okazaki, Aichi, 444-8787, Japan. kenji514@nibb.ac.jp.
authors.forename: Kenji
authors.initials: K
authors.lastname: Toyota
authors.affiliation: Okazaki Institute for Integrative Bioscience, National Institute for Basic Biology, National Institutes of Natural Sciences, Higashiyama 5-1, Myodaiji, Okazaki, Aichi, 444-8787, Japan. miyagawa@nibb.ac.jp.|Faculty of Life Science, Graduate University for Advanced Studies (SOKENDAI), 5-1 Higashiyama, Myodaiji, Okazaki, Aichi, 444-8787, Japan. miyagawa@nibb.ac.jp.
authors.forename: Shinichi
authors.initials: S
authors.lastname: Miyagawa
authors.affiliation: Department of Mathematical and Life Sciences, Graduate School of Science, Hiroshima University, Higashi-Hiroshima, 739-8526, Japan. tybig@hiroshima-u.ac.jp.
authors.forename: Takashi
authors.initials: T
authors.lastname: Yamamoto
authors.affiliation: Okazaki Institute for Integrative Bioscience, National Institute for Basic Biology, National Institutes of Natural Sciences, Higashiyama 5-1, Myodaiji, Okazaki, Aichi, 444-8787, Japan. taisen@nibb.ac.jp.|Faculty of Life Science, Graduate University for Advanced Studies (SOKENDAI), 5-1 Higashiyama, Myodaiji, Okazaki, Aichi, 444-8787, Japan. taisen@nibb.ac.jp.
authors.forename: Taisen
authors.initials: T
authors.lastname: Iguchi
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D060829:Arthropod Proteins / Q000235:genetics / Q000378:metabolism; D003621:Daphnia / Q000196:embryology / Q000235:genetics* / Q000378:metabolism; D004720:Endonucleases / Q000235:genetics / Q000378:metabolism*; D018390:Gene Targeting / Q000379:methods*; D016678:Genome; D057809:HEK293 Cells; D006801:Humans; D008845:Microinjections; D008969:Molecular Sequence Data; D016296:Mutagenesis; D059386:Reverse Genetics; D016415:Sequence Alignment; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism
chemical_list: D060829:Arthropod Proteins; D014157:Transcription Factors; D004720:Endonucleases