title: Efficient methods for targeted mutagenesis in zebrafish using zinc-finger nucleases: data from targeting of nine genes using CompoZr or CoDA ZFNs.
abstract: Recently, it has been shown that targeted mutagenesis using zinc-finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs) can be used to generate knockout zebrafish lines for analysis of their function and/or developing disease models. A number of different methods have been developed for the design and assembly of gene-specific ZFNs and TALENs, making them easily available to most zebrafish researchers. Regardless of the choice of targeting nuclease, the process of generating mutant fish is similar. It is a time-consuming and multi-step process that can benefit significantly from development of efficient high throughput methods. In this study, we used ZFNs assembled through either the CompoZr (Sigma-Aldrich) or the CoDA (context-dependent assembly) platforms to generate mutant zebrafish for nine genes. We report our improved high throughput methods for 1) evaluation of ZFNs activity by somatic lesion analysis using colony PCR, eliminating the need for plasmid DNA extractions from a large number of clones, and 2) a sensitive founder screening strategy using fluorescent PCR with PIG-tailed primers that eliminates the stutter bands and accurately identifies even single nucleotide insertions and deletions. Using these protocols, we have generated multiple mutant alleles for seven genes, five of which were targeted with CompoZr ZFNs and two with CoDA ZFNs. Our data also revealed that at least five-fold higher mRNA dose was required to achieve mutagenesis with CoDA ZFNs than with CompoZr ZFNs, and their somatic lesion frequency was lower (<5%) when compared to CopmoZr ZFNs (9-98%). This work provides high throughput protocols for efficient generation of zebrafish mutants using ZFNs and TALENs.
authors: Zebrafish Core Facility, Genetics and Molecular Biology Branch, National Human Genome Research Institute, Bethesda, Maryland, United States of America. rsood@mail.nih.gov
authors: Raman
authors: R
authors: Sood
authors: Blake
authors: B
authors: Carrington
authors: Kevin
authors: K
authors: Bishop
authors: MaryPat
authors: M
authors: Jones
authors: Alberto
authors: A
authors: Rissone
authors: Fabio
authors: F
authors: Candotti
authors: Settara C
authors: SC
authors: Chandrasekharappa
authors: Paul
authors: P
authors: Liu
mesh_terms: D000818:Animals; D001483:Base Sequence; D017931:DNA Primers; D018703:Founder Effect; D018390:Gene Targeting; D005854:Germ Cells; D006579:Heterozygote; D016296:Mutagenesis; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000096:biosynthesis; D015027:Zebrafish; D016335:Zinc Fingers
chemical_list: D017931:DNA Primers; D012333:RNA, Messenger