title: Tissue-specific and ubiquitous gene knockouts by TALEN electroporation provide new approaches to investigating gene function in Ciona.
abstract: Custom designed nucleases can simplify gene targeting experiments and have the potential to allow these techniques to be performed in a wide range of organisms. Transcriptional activator-like effector nucleases (TALENs) are starting to fulfill this potential with the advantages of low cost and fast construction times. Here, we report that TALENs are highly effective at inducing mutations in specific genomic loci in the ascidian chordate Ciona intestinalis. In Ciona there are well-established methods to introduce exogenous DNA by electroporation, and we show that this method can be used to introduce constructs that can express TALENs ubiquitously or in specific tissues. Our current protocols enable the rapid analysis of hundreds of TALEN-induced mutants. TALEN electroporations result in a high rate of mutations. These mutations can result in gene knockouts that recapitulate previously described functions of Fgf3 and Hox12. We show that TALENs can work efficiently to cause tissue-specific knockouts and demonstrate this by knocking out Hox12 in the epidermis and Fgf3 in neural tissues. We also use tissue-specific knockouts to reveal a new function of Fgf3 during ascidian larval metamorphosis.
authors.affiliation: Shimoda Marine Research Center, University of Tsukuba, Shimoda, Shizuoka, 415-0025, Japan.
authors.forename: Nicholas
authors.initials: N
authors.lastname: Treen
authors.forename: Keita
authors.initials: K
authors.lastname: Yoshida
authors.forename: Tetsushi
authors.initials: T
authors.lastname: Sakuma
authors.forename: Haruka
authors.initials: H
authors.lastname: Sasaki
authors.forename: Narudo
authors.initials: N
authors.lastname: Kawai
authors.forename: Takashi
authors.initials: T
authors.lastname: Yamamoto
authors.forename: Yasunori
authors.initials: Y
authors.lastname: Sasakura
mesh_terms: D000818:Animals; D001483:Base Sequence; D002938:Ciona intestinalis / Q000196:embryology / Q000235:genetics* / Q000378:metabolism; D004247:DNA / Q000235:genetics; D003851:Deoxyribonucleases / Q000235:genetics / Q000378:metabolism; D018274:Electroporation / Q000379:methods*; D018507:Gene Expression Regulation, Developmental; D055786:Gene Knockout Techniques / Q000379:methods*; D005818:Genetic Engineering / Q000379:methods; D008969:Molecular Sequence Data; D016296:Mutagenesis; D014018:Tissue Distribution
chemical_list: D004247:DNA; D003851:Deoxyribonucleases
keywords: Gene targeting; Reverse genetics; TALENs