RecA-mediated, targeted mutagenesis in zebrafish

• Published in: Marine biotechnology (New York, N.Y.) N.Y.), 2003-03, Vol.5 (2), p.174-184
• Main Authors: Cui, Zongbin, Yang, Ying, Kaufman, Christopher D, Agalliu, Dritan, Hackett, Perry B
• Format: Article
• Language: English
• Subjects: Animals; Animals, Genetically Modified; Cloning, Molecular; Danio rerio; DNA - genetics; DNA - metabolism; DNA Primers - genetics; DNA Probes; DNA, Recombinant - genetics; Embryos; Freshwater; Gene Expression; Genetic Engineering - methods; Genetic Vectors; Green Fluorescent Proteins; Irradiation; Luminescent Proteins - genetics; Marine biology; Mutagenesis; Mutagenesis, Site-Directed; Rec A Recombinases - metabolism; Studies; Ultraviolet radiation; Zebrafish; Zebrafish - embryology; Zebrafish - genetics
• ISSN: 1436-2228; 1436-2236
• DOI: 10.1007/s10126-002-0059-0

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.