Efficient methods for targeted mutagenesis in zebrafish using zinc-finger nucleases: data from targeting of nine genes using CompoZr or CoDA ZFNs

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 b...

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Bibliographic Details
Published in:PloS one 2013-02, Vol.8 (2), p.e57239
Main Authors: Sood, Raman, Carrington, Blake, Bishop, Kevin, Jones, MaryPat, Rissone, Alberto, Candotti, Fabio, Chandrasekharappa, Settara C, Liu, Paul
Format: Article
Language:English
Subjects:
Analysis
Animal genetic engineering
Animal models
Animals
Assembly
Base Sequence
Biology
Biotechnology
Cloning
Danio rerio
Deoxyribonucleic acid
DNA
DNA binding proteins
DNA Primers
Fluorescence
Founder Effect
Gene mutation
Gene Targeting
Genes
Genetic engineering
Genomes
Genomics
Genotype & phenotype
Germ Cells
Heterozygote
Laboratories
Molecular biology
Mutagenesis
Mutants
Mutation
Nuclease
Nucleases
Plasmids
Polymerase Chain Reaction
Primers
Proteins
RNA
RNA, Messenger - biosynthesis
Site-directed mutagenesis
Sperm
Targeted mutagenesis
Transcription
Transcription activator-like effector nucleases
Zebrafish
Zinc
Zinc finger proteins
Zinc Fingers
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Summary: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 (
ISSN:1932-6203
1932-6203
DOI:10.1371/journal.pone.0057239