RecA-mediated, targeted mutagenesis in zebrafish

Zongbin Cui, Ying Yang, Christopher D. Kaufman, Dritan Agalliu, Perry B. Hackett

Research output: Contribution to journalArticlepeer-review

19 Scopus citations

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.

Original languageEnglish (US)
Pages (from-to)174-184
Number of pages11
JournalMarine Biotechnology
Volume5
Issue number2
DOIs
StatePublished - Mar 2003

Keywords

  • Gene targeting
  • RecA protein
  • Site-specific mutagenesis
  • Zebrafish

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