TY - JOUR
T1 - Targeted gene disruption by use of transcription activator-like effector nuclease (TALEN) in the water flea Daphnia pulex
AU - Hiruta, Chizue
AU - Ogino, Yukiko
AU - Sakuma, Tetsushi
AU - Toyota, Kenji
AU - Miyagawa, Shinichi
AU - Yamamoto, Takashi
AU - Iguchi, Taisen
N1 - Funding Information:
Daphnia pulex genomic sequence data were produced by The Center for Genomics and Bioinformatics at Indiana University and distributed via wFleaBase in collaboration with the Daphnia Genomics Consortium https:// wiki.cgb.indiana.edu/display/DGC/Home. We thank Brenna Doheny for reviewing and editing the manuscript; members of the Iguchi laboratory for helpful advice and discussions; members of Yamamoto’s laboratory for assistance in TALEN construction. This work was supported by a JSPS Research Fellowship for Young Scientists to CH (No.12J04065), KT (No.12J05579), JSPS KAKENHI Grant Number 26840115 to CH, a grant from Ministry of the Environment, Japan and MEXT KAKENHI Grant Numbers 24657022, 24370029 to TI. Our work benefits from, and contributes to, the Daphnia Genomics Consortium.
Publisher Copyright:
© 2014 Hiruta et al.
PY - 2014
Y1 - 2014
N2 - 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.
AB - 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.
UR - http://www.scopus.com/inward/record.url?scp=84941269676&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=84941269676&partnerID=8YFLogxK
U2 - 10.1186/s12896-014-0095-7
DO - 10.1186/s12896-014-0095-7
M3 - Article
C2 - 25404042
AN - SCOPUS:84941269676
SN - 1472-6750
VL - 14
SP - 1
EP - 8
JO - BMC Biotechnology
JF - BMC Biotechnology
IS - 1
M1 - 95
ER -
