Targeted gene disruption by use of transcription activator-like effector nuclease (TALEN) in the water flea Daphnia pulex

BMC Biotechnology

Chizue Hiruta¹  Yukiko Ogino³  Tetsushi Sakuma²  Kenji Toyota³  Shinichi Miyagawa³  Takashi Yamamoto²  Taisen Iguchi³ 

[1] Okazaki Institute for Integrative Bioscience, National Institute for Basic Biology, National Institutes of Natural Sciences, Higashiyama 5-1, Myodaiji, Okazaki 444-8787, Aichi, Japan

[2] Department of Mathematical and Life Sciences, Graduate School of Science, Hiroshima University, Higashi-Hiroshima 739-8526, Japan

[3] Faculty of Life Science, Graduate University for Advanced Studies (SOKENDAI), 5-1 Higashiyama, Myodaiji, Okazaki 444-8787, Aichi, Japan

关键词: Genome editing;    Gene manipulation;    Targeted mutagenesis;    Knock-out;    Gene disruption;    Platinum TALEN;    Distal-less;    Daphnia pulex;   

Others  :  1084439
DOI  :  10.1186/s12896-014-0095-7

 received in 2014-07-26, accepted in 2014-10-28,  发布年份 2014

【 摘 要 】

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.

【 授权许可】

   
2014 Hiruta et al.; licensee BioMed Central Ltd.

【 图 表 】

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