RecA-mediated, targeted mutagenesis in zebrafish.
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.
Cui
Zongbin
Z
Department of Genetics, Cell Biology and Development and The Arnold and Mabel Beckman Center for Transposon Research, University of Minnesota, St. Paul, MN 55108-1095, USA.
Yang
Ying
Y
Kaufman
Christopher D
CD
Agalliu
Dritan
D
Hackett
Perry B
PB
D000818:Animals; D030801:Animals, Genetically Modified; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics / Q000378:metabolism*; D017931:DNA Primers / Q000235:genetics; D015342:DNA Probes; D004274:DNA, Recombinant / Q000235:genetics; D015870:Gene Expression; D005818:Genetic Engineering / Q000379:methods; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins / Q000235:genetics*; D016297:Mutagenesis, Site-Directed; D011938:Rec A Recombinases / Q000378:metabolism*; D015027:Zebrafish / Q000196:embryology / Q000235:genetics*
D017931:DNA Primers; D015342:DNA Probes; D004274:DNA, Recombinant; D008164:Luminescent Proteins; D049452:Green Fluorescent Proteins; D004247:DNA; D011938:Rec A Recombinases