PUBLICATION
RecA-mediated, targeted mutagenesis in zebrafish
- Authors
- Cui, Z., Yang, Y., Kaufman, C.D., Agalliu, D., and Hackett, P.B.
- ID
- ZDB-PUB-030728-16
- Date
- 2003
- Source
- Marine biotechnology (New York, N.Y.) 5(2): 174-184 (Journal)
- Registered Authors
- Cui, Zongbin, Hackett, Perry B., Kaufman, Christopher
- Keywords
- none
- MeSH Terms
-
- Animals
- Animals, Genetically Modified
- Cloning, Molecular
- DNA/genetics
- DNA/metabolism*
- DNA Primers/genetics
- DNA Probes
- DNA, Recombinant/genetics
- Gene Expression*
- Genetic Engineering/methods
- Genetic Vectors
- Green Fluorescent Proteins
- Luminescent Proteins/genetics*
- Mutagenesis, Site-Directed*
- Rec A Recombinases/metabolism*
- Zebrafish/embryology
- Zebrafish/genetics*
- PubMed
- 12876654 Full text @ Mar. Biotechnol.
Citation
Cui, Z., Yang, Y., Kaufman, C.D., Agalliu, D., and Hackett, P.B. (2003) RecA-mediated, targeted mutagenesis in zebrafish. Marine biotechnology (New York, N.Y.). 5(2):174-184.
Abstract
We have evaluated the efficacy of RecA, a prokaryotic protein involved with homologous recombination, to direct site-specific mutagenesis in zebrafish embryos. For this we coinjected a vector containing a mutated enhanced green fluorescent protein (EGFP) gene plus 236-nucleotide corrective single-stranded DNAs coated with RecA into 1-cell zebrafish embryos. Twenty-hours after fertilization, about 5% to 20% of injected embryos showed EGFP expression in 1 or more cells when RecA-coated corrective DNAs were used, but not when RecA was omitted. Mutated EGFP genes with 1-bp insertions or deletions were inefficiently activated, whereas those with 7-bp insertions were activated about 4-fold more efficiently. RecA-coated template strand had a higher efficiency than its complementary strand in activation of EGFP expression. Prior irradiation of the embryos with UV light enhanced RecA-mediated restoration of gene activity, suggesting that the effects we observed were augmented by one or more factors of zebrafish DNA repair systems.
Genes / Markers
Expression
Phenotype
Mutations / Transgenics
Human Disease / Model
Sequence Targeting Reagents
Fish
Orthology
Engineered Foreign Genes
Mapping