title: Bleomycin stimulates targeted gene repair directed by single-stranded oligodeoxynucleotides in BHK-21 cells.
abstract: Synthetic single-stranded oligodeoxynucleotides (ssODNs) designed for single nucleotide mismatch can induce targeted gene repair (TGR) in chromosomal DNA. TGR directed by ssODNs is an ideal method for gene manipulation because it precisely exchanges single nucleotides in targeted DNA without additional modification to the genome. However, TGR efficiency is still insufficient for practical use. Here, we report that bleomycin (BLM), which breaks double-stranded DNAs, stimulates TGR directed by ssODNs in BHK-21 cells. To evaluate TGR efficiency, transformants of BHK-21 cells permanently expressing mutant EGFP genes were established. Microscopy and restriction fragment length polymorphism (RFLP) analysis clearly showed that the mutant EGFP gene was restored to a normal EGFP gene with an ssODN comple-mentary to a transcribed strand. TGR efficiency was measured by counting the number of GFP-fluorescent cells with a flow cytometer. TGR efficiency of the ssODN complementary to a transcribed strand was significantly higher than that of ssODN complementary to a non-transcribed strand, indicating that a strand bias existed. Exposure of cells to BLM stimulated TGR directed by ssODN in a dose-dependent manner, and high concentrations of BLM (750 ng/ml) abolished strand bias on TGR efficiency. These results suggest that BLM stimulates TGR directed by ssODNs in BHK-21 cells, probably due to activation of the DNA repair system.
authors.affiliation: Laboratory of Animal Nutrition, Department of Applied Molecular Biosciences, Graduate School of Bioagricultural Sciences, Nagoya University, Nagoya 464-8601, Japan. i043006d@mbox.nagoya-u.ac.jp
authors.forename: Takayuki
authors.initials: T
authors.lastname: Suzuki
authors.forename: Atsushi
authors.initials: A
authors.lastname: Murai
authors.forename: Tatsuo
authors.initials: T
authors.lastname: Muramatsu
mesh_terms: D000818:Animals; D000903:Antibiotics, Antineoplastic / Q000494:pharmacology; D001483:Base Sequence; D001761:Bleomycin / Q000494:pharmacology*; D015139:Blotting, Southern; D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects / Q000235:genetics; D004247:DNA / Q000235:genetics / Q000378:metabolism; D004260:DNA Repair / Q000187:drug effects* / Q000235:genetics; D004277:DNA, Single-Stranded / Q000235:genetics*; D015246:Deoxyribonuclease EcoRI / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D005434:Flow Cytometry; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides / Q000235:genetics*; D010957:Plasmids / Q000235:genetics; D017354:Point Mutation; D012150:Polymorphism, Restriction Fragment Length; D014162:Transfection
chemical_list: D000903:Antibiotics, Antineoplastic; D004277:DNA, Single-Stranded; D009838:Oligodeoxyribonucleotides; D001761:Bleomycin; D049452:Green Fluorescent Proteins; D004247:DNA; D015246:Deoxyribonuclease EcoRI