TITLE

Efficient Methods for Targeted Mutagenesis in Zebrafish Using Zinc-Finger Nucleases: Data from Targeting of Nine Genes Using CompoZr or CoDA ZFNs

AUTHOR(S)
Sood, Raman; Carrington, Blake; Bishop, Kevin; Jones, MaryPat; Rissone, Alberto; Candotti, Fabio; Chandrasekharappa, Settara C.; Liu, Paul
PUB. DATE
February 2013
SOURCE
PLoS ONE;Feb2013, Vol. 8 Issue 2, p1
SOURCE TYPE
Academic Journal
DOC. TYPE
Article
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.
ACCESSION #
87625313

 

Related Articles

  • Tumor-specific signaling to p53 is mimicked by Mdm2 inactivation in zebrafish: insights from mdm2 and mdm4 mutant zebrafish. Chua, J S; Liew, H P; Guo, L; Lane, D P // Oncogene;11/26/2015, Vol. 34 Issue 48, p5933 

    In mice, the deletion of either Mdm2 or Mdm4 results in a p53-dependent embryonic lethality. We used zinc-finger nucleases to construct mutations in the mdm2 and mdm4 genes of zebrafish. Although the loss of mdm2 results in a p53-dependent early embryonic lethality, mdm4 mutant fish are viable...

  • Biotechnology: Genetic dexterity.  // Nature;6/5/2008, Vol. 453 Issue 7196, p700 

    The article reports on the gene selection method developed by two groups of research teams in the U.S. Researchers adopted chimaeric enzymes called zinc-finger nucleases to disrupt selected genes of the zebrafish which they chose as a laboratory model. Sharon Amacher and colleagues at the...

  • High Efficiency In Vivo Genome Engineering with a Simplified 15-RVD GoldyTALEN Design Ma, Alvin C.; Lee, Han B.; Clark, Karl J.; Ekker, Stephen C. // PLoS ONE;May2013, Vol. 8 Issue 5, p1 

    Transcription activator-like effector nucleases (TALENs) enable genome engineering in cell culture and many organisms. Recently, the GoldyTALEN scaffold was shown to readily introduce mutations in zebrafish (Danio rerio) and livestock through non-homologous end joining (NHEJ) and...

  • Emerging gene knockout technology in zebrafish: zinc-finger nucleases. Amacher, Sharon L. // Briefings in Functional Genomics & Proteomics;Nov2008, Vol. 7 Issue 6, p460 

    One advantage of the zebrafish model system is the ability to use forward genetics to reveal critical gene functions by their mutant phenotype. Reverse genetic tools are available, although, it is more challenging and time-consuming to identify mutations in specific genes of interest and...

  • A Designed Zinc-finger Transcriptional Repressor of Phospholamban Improves Function of the Failing Heart. Zhang, H Steve; Liu, Dingang; Huang, Yan; Schmidt, Stefan; Hickey, Reed; Guschin, Dmitry; Su, Haili; Jovin, Ion S; Kunis, Mike; Hinkley, Sarah; Liang, Yuxin; Hinh, Linda; Spratt, S Kaye; Case, Casey C; Rebar, Edward J; Ehrlich, Barbara; Gregory, Philip D; Giordano, Frank J // Molecular Therapy;Aug2012, Vol. 20 Issue 8, p1508 

    Selective inhibition of disease-related proteins underpins the majority of successful drug-target interactions. However, development of effective antagonists is often hampered by targets that are not druggable using conventional approaches. Here, we apply engineered zinc-finger protein...

  • Progress and Problems with Viral Vectors for Delivery of Talens. Bergmann, Thorsten; Schulz, Eric; Ehrhardt, Anja // Journal of Molecular & Genetic Medicine (Library Publishing Medi;2014, Vol. 8 Issue 1, p1 

    It has long been envisaged that gene disruption or gene correction in affected target cells can be efficiently conducted in vitro and in vivo and over the recent years several tools for achieving this goal were developed. Designer nucleases such as zinc finger nucleases (ZFNs) were extensively...

  • Magnetic Separation and Antibiotics Selection Enable Enrichment of Cells with ZFN/TALEN-Induced Mutations. Kim, Hyojin; Kim, Myung-Sun; Wee, Gabbine; Lee, Choong-il; Kim, Hyongbum; Kim, Jin-Soo // PLoS ONE;Feb2013, Vol. 8 Issue 2, p1 

    The ability to enrich cells with targeted mutations greatly facilitates the process of using engineered nucleases, including zinc-finger nucleases and transcription activator-like effector nucleases, to construct such cells. We previously used surrogate reporters to enrich cells containing...

  • Identification of Zfp521/ZNF521 as a cooperative gene for E2A-HLF to develop acute B-lineage leukemia. Yamasaki, N.; Miyazaki, K.; Nagamachi, A.; Koller, R.; Oda, H.; Miyazaki, M.; Sasaki, T.; Honda, Z.-i; Wolff, L.; Inaba, T.; Honda, H. // Oncogene;4/1/2010, Vol. 29 Issue 13, p1963 

    E2A-hepatic leukemia factor (HLF) is a chimeric protein found in B-lineage acute lymphoblastic leukemia (ALL) with t(17;19). To analyze the leukemogenic process and to create model mice for t(17;19)-positive leukemia, we generated inducible knock-in (iKI) mice for E2A-HLF. Despite the induced...

  • Nr2f1b control venous specification and angiogenic patterning during zebrafish vascular development. Ru-Fang Li; Ting-Yun Wu; Yu-Zheng Mou; Yi-Shan Wang; Chun-Lin Chen; Chang-Yi Wu // Journal of Biomedical Science;11/17/2015, Vol. 22, p1 

    Background: The specification of vein and the patterning of intersegmental vessels (ISV) controlled by transcription factor is not fully characterized. The orphan nuclear receptor Chicken ovalbumin upstream promoter transcription factor II (CoupTFII, a.k.a NR2F2) positively regulates vein...

Share

Read the Article

Courtesy of VIRGINIA BEACH PUBLIC LIBRARY AND SYSTEM

Sorry, but this item is not currently available from your library.

Try another library?
Sign out of this library

Other Topics