title: TALEN-mediated homologous recombination in Daphnia magna.
abstract: Transcription Activator-Like Effector Nucleases (TALENs) offer versatile tools to engineer endogenous genomic loci in various organisms. We established a homologous recombination (HR)-based knock-in using TALEN in the crustacean Daphnia magna, a model for ecological and toxicological genomics. We constructed TALENs and designed the 67 bp donor insert targeting a point deletion in the eyeless mutant that shows eye deformities. Co-injection of the TALEN mRNA with donor DNA into eggs led to the precise integration of the donor insert in the germ line, which recovered eye deformities in offspring. The frequency of HR events in the germ line was 2% by using both plasmid and single strand oligo DNA with 1.5 kb and 80 nt homology to the target. Deficiency of ligase 4 involved in non-homologous end joining repair did not increase the HR efficiency. Our data represent efficient HR-based knock-in by TALENs in D. magna, which is a promising tool to understand Daphnia gene functions.
authors.affiliation: Department of Biotechnology, Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka, Japan.
authors.forename: Takashi
authors.initials: T
authors.lastname: Nakanishi
authors.affiliation: Department of Biotechnology, Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka, Japan.|Frontier Research Base for Global Young Researchers, Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka, Japan.
authors.forename: Yasuhiko
authors.initials: Y
authors.lastname: Kato
authors.affiliation: Department of Biotechnology, Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka, Japan.
authors.forename: Tomoaki
authors.initials: T
authors.lastname: Matsuura
authors.affiliation: Department of Biotechnology, Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka, Japan.
authors.forename: Hajime
authors.initials: H
authors.lastname: Watanabe
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D004247:DNA / Q000235:genetics / Q000378:metabolism; D011088:DNA Ligases / Q000172:deficiency / Q000235:genetics; D004262:DNA Restriction Enzymes / Q000235:genetics* / Q000378:metabolism; D003621:Daphnia / Q000235:genetics* / Q000378:metabolism; D005124:Eye Abnormalities / Q000235:genetics; D055879:Gene Knock-In Techniques / Q000379:methods; D005818:Genetic Engineering / Q000379:methods*; D005854:Germ Cells / Q000378:metabolism; D059765:Homologous Recombination; D008957:Models, Genetic; D008969:Molecular Sequence Data; D009154:Mutation; D010641:Phenotype; D015203:Reproducibility of Results
chemical_list: D004247:DNA; D004262:DNA Restriction Enzymes; D011088:DNA Ligases