Characterization of zebrafish Rad52 and replication protein A for oligonucleotide-mediated mutagenesis.
Zebrafish has become a favorite model organism not only in genetics and developmental biology, but also for the study of cancer, neuroscience and metabolism. However, strategies for reverse genetics in zebrafish are mostly limited to the use of antisense oligonucleotides, and therefore the development of other targeting methods is highly desirable. Here, we report an approach to gene targeting in this system in which single-stranded oligonucleotides and zebrafish Rad52 protein are employed. It has been proposed that a single-stranded oligonucleotide containing a mutation can be incorporated into the genome by annealing to the single-stranded region of the lagging strand of the replication fork. Rad52 is expected to accelerate the annealing step. In vitro experiments using purified truncated Rad52 proteins and replication protein A (RPA) showed that annealing of oligonucleotides is accelerated by Rad52 in the presence of RPA. We developed a simple and sensitive PCR-based method to detect point mutations in the genome. In exploratory experiments, we found that microinjection of single-stranded oligonucleotide targeted to a specific gene together with truncated Rad52 into zebrafish embryos resulted in a low level of recombinant copies in 3 of the 80 embryos tested under these conditions.
Takahashi
Nobuhiro
N
Laboratory of Molecular Genetics, National Institute of Child Health and Human Development, National Institutes of Health Bethesda, MD 20892-2790, USA.
Dawid
Igor B
IB
D000595:Amino Acid Sequence; D000818:Animals; D002478:Cells, Cultured; D003001:Cloning, Molecular; D004268:DNA-Binding Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D008845:Microinjections; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D019913:Nuclear Localization Signals; D009841:Oligonucleotides / Q000008:administration & dosage / Q000737:chemistry* / Q000378:metabolism; D017354:Point Mutation; D016133:Polymerase Chain Reaction; D051721:Rad52 DNA Repair and Recombination Protein; D051716:Replication Protein A; D016415:Sequence Alignment; D015027:Zebrafish / Q000196:embryology* / Q000235:genetics; D029961:Zebrafish Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*
D004268:DNA-Binding Proteins; D019913:Nuclear Localization Signals; D009841:Oligonucleotides; D051721:Rad52 DNA Repair and Recombination Protein; C502793:Rad52 protein, zebrafish; D051716:Replication Protein A; D029961:Zebrafish Proteins