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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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Autores principales: Sood, Raman, Carrington, Blake, Bishop, Kevin, Jones, MaryPat, Rissone, Alberto, Candotti, Fabio, Chandrasekharappa, Settara C., Liu, Paul
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Public Library of Science 2013
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3579846/
https://www.ncbi.nlm.nih.gov/pubmed/23451191
http://dx.doi.org/10.1371/journal.pone.0057239
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author Sood, Raman
Carrington, Blake
Bishop, Kevin
Jones, MaryPat
Rissone, Alberto
Candotti, Fabio
Chandrasekharappa, Settara C.
Liu, Paul
author_facet Sood, Raman
Carrington, Blake
Bishop, Kevin
Jones, MaryPat
Rissone, Alberto
Candotti, Fabio
Chandrasekharappa, Settara C.
Liu, Paul
author_sort Sood, Raman
collection PubMed
description 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.
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spelling pubmed-35798462013-02-28 Efficient Methods for Targeted Mutagenesis in Zebrafish Using Zinc-Finger Nucleases: Data from Targeting of Nine Genes Using CompoZr or CoDA ZFNs Sood, Raman Carrington, Blake Bishop, Kevin Jones, MaryPat Rissone, Alberto Candotti, Fabio Chandrasekharappa, Settara C. Liu, Paul PLoS One Research Article 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. Public Library of Science 2013-02-22 /pmc/articles/PMC3579846/ /pubmed/23451191 http://dx.doi.org/10.1371/journal.pone.0057239 Text en https://creativecommons.org/publicdomain/zero/1.0/ This is an open-access article distributed under the terms of the Creative Commons Public Domain declaration, which stipulates that, once placed in the public domain, this work may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose.
spellingShingle Research Article
Sood, Raman
Carrington, Blake
Bishop, Kevin
Jones, MaryPat
Rissone, Alberto
Candotti, Fabio
Chandrasekharappa, Settara C.
Liu, Paul
Efficient Methods for Targeted Mutagenesis in Zebrafish Using Zinc-Finger Nucleases: Data from Targeting of Nine Genes Using CompoZr or CoDA ZFNs
title Efficient Methods for Targeted Mutagenesis in Zebrafish Using Zinc-Finger Nucleases: Data from Targeting of Nine Genes Using CompoZr or CoDA ZFNs
title_full Efficient Methods for Targeted Mutagenesis in Zebrafish Using Zinc-Finger Nucleases: Data from Targeting of Nine Genes Using CompoZr or CoDA ZFNs
title_fullStr Efficient Methods for Targeted Mutagenesis in Zebrafish Using Zinc-Finger Nucleases: Data from Targeting of Nine Genes Using CompoZr or CoDA ZFNs
title_full_unstemmed Efficient Methods for Targeted Mutagenesis in Zebrafish Using Zinc-Finger Nucleases: Data from Targeting of Nine Genes Using CompoZr or CoDA ZFNs
title_short Efficient Methods for Targeted Mutagenesis in Zebrafish Using Zinc-Finger Nucleases: Data from Targeting of Nine Genes Using CompoZr or CoDA ZFNs
title_sort efficient methods for targeted mutagenesis in zebrafish using zinc-finger nucleases: data from targeting of nine genes using compozr or coda zfns
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3579846/
https://www.ncbi.nlm.nih.gov/pubmed/23451191
http://dx.doi.org/10.1371/journal.pone.0057239
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