title: Methods for targeted mutagenesis in zebrafish using TALENs.
abstract: The transcription activator-like effector (TALE) nucleases, or TALENs, are customizable restriction enzymes that may be used to induce mutations at nearly any investigator-specified DNA sequence in zebrafish. The DNA-binding specificities of TALENs are determined by a protein array comprised of four types of TALE repeats, where each repeat recognizes a different DNA base. Here, we describe methods for constructing TALEN vectors that have been shown to achieve high success rates and mutation efficiencies in zebrafish. In addition, we discuss simple techniques and protocols that can be used to detect TALEN-induced mutations at almost any genomic locus. These methods should enable zebrafish researchers to quickly generate targeted mutations at their genes-of-interest.
authors.affiliation: Cardiovascular Research Center, Massachusetts General Hospital, 149 13th street, Room 4201, Charlestown, MA 02129, USA; Department of Medicine, Harvard Medical School, 25 Shattuck St, Boston, MA 02115, USA.
authors.forename: Woong Y
authors.initials: WY
authors.lastname: Hwang
authors.affiliation: Cardiovascular Research Center, Massachusetts General Hospital, 149 13th street, Room 4201, Charlestown, MA 02129, USA; Department of Medicine, Harvard Medical School, 25 Shattuck St, Boston, MA 02115, USA; Broad Institute, 7 Cambridge Center, Cambridge, MA 02142, USA.
authors.forename: Randall T
authors.initials: RT
authors.lastname: Peterson
authors.affiliation: Cardiovascular Research Center, Massachusetts General Hospital, 149 13th street, Room 4201, Charlestown, MA 02129, USA; Department of Medicine, Harvard Medical School, 25 Shattuck St, Boston, MA 02115, USA. Electronic address: JYEH1@mgh.harvard.edu.
authors.forename: Jing-Ruey Joanna
authors.initials: JR
authors.lastname: Yeh
mesh_terms: D000818:Animals; D003851:Deoxyribonucleases / Q000737:chemistry / Q000235:genetics; D016297:Mutagenesis, Site-Directed / Q000379:methods*; D015202:Protein Engineering; D015027:Zebrafish / Q000235:genetics*
chemical_list: D003851:Deoxyribonucleases
keywords: Gene disruption; Gene targeting; Genome engineering; Knockout; Site-specific nuclease; Transcription activator-like effector nuclease; Zebrafish
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