Abstract
BackgroundThe 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.ResultsWe 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.ConclusionsWe 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.Electronic supplementary materialThe online version of this article (doi:10.1186/s12896-014-0095-7) contains supplementary material, which is available to authorized users.
Highlights
The cosmopolitan microcrustacean Daphnia pulex provides a model system for both human health research and monitoring ecosystem integrity
We have evaluated the transcription activator-like effector nuclease (TALEN) activities using human cells for various animal applications including sea urchin [29] and fruit fly [27,30], suggesting that the activity score measured in human cells is, in principle, a good indication of the level of inducing double-strand breaks (DSBs) in a wide range of animals
single-strand annealing (SSA) assay can directly be performed with the constructed TALEN plasmids in human cells, so that we can know the quality of the constructed TALENs before making mRNAs and injecting them into the animals
Summary
The cosmopolitan microcrustacean Daphnia pulex provides a model system for both human health research and monitoring ecosystem integrity. The D. pulex genome possesses as many as about 31,000 genes; this large suite of genes may provide the arsenal responsible for the organism’s responsiveness to environmental challenges [3]. There are no effective methods for manipulating genes, and only transient analysis of gene function by the RNA interference (RNAi) method [6] is available. The RNAi system in D. pulex suffers from several weaknesses, including incomplete silencing, transient effects, and limited analyzable stages. Because of these situations, even in the post-genomic era, the establishment of a gene manipulation technique has been eagerly anticipated to address the characterization of gene function in D. pulex
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
