Single-strand DNA-mediated targeted mutagenesis of genomic DNA in early mouse embryos is stimulated by Rad51/54 and by Ku70/86 inhibition.
Low and variable efficiency is a major problem in targeted gene alteration, which is used as a primary tool in gene therapy and animal model studies. We tested several types of constructs alone, or in combination with other factors, to introduce a point mutation into the alphaB-crystallin gene in one-celled mouse embryos. We found that co-injection of ssDNA along with antibodies against Ku70/86, or supplementing the system with hRad51/hRad54, increases efficiency of targeted mutagenesis. These findings suggest that proteins in the homologous recombination DNA repair pathway contribute, and that proteins involved in the alternative nonhomologous end-joining pathway inhibit, ssDNA-mediated targeted mutagenesis. This is the first successful demonstration of targeted mutation in early mouse embryos. This novel methodology of supplying protein factors to stimulate gene modification in the nucleus has not been previously reported.
Morozov
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Laboratory of Molecular and Developmental Biology, National Eye Institute, National Institutes of Health, DHHS, Bethesda, MD, USA. morozovv@nei.nih.gov
Wawrousek
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D000818:Animals; D000906:Antibodies / Q000008:administration & dosage; D034961:Antigens, Nuclear / Q000276:immunology; D002970:Cleavage Stage, Ovum / Q000378:metabolism*; D004277:DNA, Single-Stranded / Q000008:administration & dosage*; D004268:DNA-Binding Proteins / Q000276:immunology; D005260:Female; D018390:Gene Targeting; D015316:Genetic Therapy / Q000379:methods*; D016678:Genome; D006801:Humans; D000072200:Ku Autoantigen; D051379:Mice; D008845:Microinjections; D016296:Mutagenesis; D017354:Point Mutation; D012150:Polymorphism, Restriction Fragment Length; D051135:Rad51 Recombinase / Q000008:administration & dosage; D038204:beta-Crystallins / Q000235:genetics*
D000906:Antibodies; D034961:Antigens, Nuclear; D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; D038204:beta-Crystallins; D051135:Rad51 Recombinase; C000606509:Xrcc6 protein, human; C000606510:Xrcc6 protein, mouse; D000072200:Ku Autoantigen