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AAV-mediated gene targeting methods for human cells

Nature Protocols volume 6, pages 482–501 (2011)Cite this article

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Abstract

Gene targeting with adeno-associated virus (AAV) vectors has been demonstrated in multiple human cell types, with targeting frequencies ranging from 10−5 to 10−2 per infected cell. These targeting frequencies are 1–4 logs higher than those obtained by conventional transfection or electroporation approaches. A wide variety of different types of mutations can be introduced into chromosomal loci with high fidelity and without genotoxicity. Here we provide a detailed protocol for gene targeting in human cells with AAV vectors. We describe methods for vector design, stock preparation and titration. Optimized transduction protocols are provided for human pluripotent stem cells, mesenchymal stem cells, fibroblasts and transformed cell lines, as well as a method for identifying targeted clones by Southern blots. This protocol (from vector design through a single round of targeting and screening) can be completed in ∼10 weeks; each subsequent round of targeting and screening should take an additional 7 weeks.

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Figure 1: Vector design strategies.
Figure 2: Detecting homologous recombination with a Southern blot.

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Acknowledgements

This work was supported by grants DK55759, HL53750 AR48328 and GM086497 to D.W.R. from the US National Institutes of Health. We thank D.R. Deyle for helpful suggestions.

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Authors and Affiliations

  1. Department of Medicine, University of Washington, Seattle, WA, USA

    Iram F Khan, Roli K Hirata & David W Russell

  2. Department of Biochemistry, University of Washington, Seattle, WA, USA

    David W Russell

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  1. Iram F Khan
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I.F.K., R.K.H. and D.W.R. wrote the manuscript.

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Correspondence to David W Russell.

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I.F.K. and R.K.H have no competing financial interests. D.W.R. is on the Scientific Advisory Board of Horizon Discovery Limited.

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Khan, I., Hirata, R. & Russell, D. AAV-mediated gene targeting methods for human cells. Nat Protoc 6, 482–501 (2011). https://doi.org/10.1038/nprot.2011.301

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