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Delivery and mechanistic considerations for the production of knock-in mice by single-stranded oligonucleotide gene targeting

Gene Therapy volume 14, pages 304–315 (2007)Cite this article

Abstract

Single-stranded oligodeoxynucleotide (ssODN) gene targeting may facilitate animal model creation and gene repair therapy. Lipofection of ssODN can introduce point mutations into target genes. However, typical efficiencies in mouse embryonic stem cells (ESC) are <10−4, leaving corrections too rare to effectively identify. We developed ESC lines with an integrated mutant neomycin resistance gene (Tyr22Ter). After targeting with ssODN, repaired cells survive selection in G418. Correction efficiencies varied with different lipofection procedures, clonal lines, and ssODN designs, ranging from 1 to 100 corrections per million cells plated. Uptake studies using cell sorting of Cy5-labelled ssODN showed 40% of the corrections concentrated in the best transfected 22% of cells. Four different basepair mismatches were tested and results show that the base-specificity of the mismatch is critical. Dual mismatch ssODN also showed mismatch preferences. These ESC lines may facilitate development of improved ssODN targeting technologies for either animal production or ex vivo gene therapy.

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Acknowledgements

The assistance of Richard Schretzenmair and Bill Murphy of the University of Pennsylvania Flow Cytometry and Cell Sorting Facility is gratefully acknowledged. We thank Gui-shuang Ying and Chengcheng Liu of the Center of Preventive Ophthalmology and Biostatistics for assistance with statistical analysis. This work was funded by Grants from NIH HL66565 (SLD), NIH PO1-CA72765, the Rosanne Silbermann Foundation, the Mackall Foundation Trust and the FM Kirby Foundation (EAP).

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Authors and Affiliations

  1. Department of Chemical and Biomolecular Engineering, Institute for Medicine and Engineering, University of Pennsylvania, Philadelphia, PA, USA

    B R Murphy & S L Diamond

  2. FM Kirby Center for Molecular Ophthalmology, Scheie Eye Institute, 305 Stellar-Chance Laboratory, University of Pennsylvania, Philadelphia, PA, USA

    H S Moayedpardazi & E A Pierce

  3. Department of Medicine, Hematology/Oncology, Abramson Cancer Center, University of Pennsylvania, Philadelphia, PA, USA

    A M Gewirtz

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  1. B R Murphy
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  2. H S Moayedpardazi
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  4. S L Diamond
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  5. E A Pierce
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Correspondence to E A Pierce.

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Murphy, B., Moayedpardazi, H., Gewirtz, A. et al. Delivery and mechanistic considerations for the production of knock-in mice by single-stranded oligonucleotide gene targeting. Gene Ther 14, 304–315 (2007). https://doi.org/10.1038/sj.gt.3302866

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