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Single-strand DNA-mediated targeted mutagenesis of genomic DNA in early mouse embryos is stimulated by Rad51/54 and by Ku70/86 inhibition

Gene Therapy volume 15, pages 468–472 (2008)Cite this article

Abstract

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 αB-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.

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Acknowledgements

This research was supported by the Intramural Research Program of the NEI, NIH, DHHS. We thank Dr Patrick Sung, Yale University School of Medicine for kindly providing us with recombinant human Rad51, Rad51KR and Rad54 proteins. We also thank Dr Joram Piatigorsky from NEI, NIH, for helpful discussions, critical reading of the manuscript and support of this project.

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  1. Laboratory of Molecular and Developmental Biology, National Eye Institute, National Institutes of Health, DHHS, Bethesda, MD, USA

    V Morozov & E F Wawrousek

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  1. V Morozov
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  2. E F Wawrousek
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Correspondence to V Morozov.

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Supplementary Information accompanies the paper on Gene Therapy website (http://www.nature.com/gt)

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Morozov, V., Wawrousek, E. Single-strand DNA-mediated targeted mutagenesis of genomic DNA in early mouse embryos is stimulated by Rad51/54 and by Ku70/86 inhibition. Gene Ther 15, 468–472 (2008). https://doi.org/10.1038/sj.gt.3303088

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