ZFIN ID: ZDB-PUB-030728-16
RecA-mediated, targeted mutagenesis in zebrafish
Cui, Z., Yang, Y., Kaufman, C.D., Agalliu, D., and Hackett, P.B.
Date: 2003
Source: Mar. Biotechnol. 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.
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.
ADDITIONAL INFORMATIONNo data available