Abstract
The ability to edit the genome of cell lines has provided valuable insights into biological processes and the contribution of specific mutations to disease biology. These techniques fall into two categories based on the DNA repair mechanism that is used to incorporate the genetic change. Nuclease-based technologies, such as Zinc-Finger Nucleases, TALENS, and Crispr/Cas9, rely on non-homologous end-joining (NHEJ) and homology directed repair (HDR) to generate a range of genetic modifications. Adeno-Associated Virus (AAV) utilizes homologous recombination to generate precise and predictable genetic modifications directly at the target locus. AAV has been used to create over 500 human isogenic cell lines comprising a wide range of genetic alterations from gene knockouts, insertions of point mutations, indels, epitope tags, and reporter genes. Here we describe the generation and use of AAV gene targeting vectors and viruses to create targeted isogenic cell lines.
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An up to date list of isogenic cell lines can be found at: http://www.horizondiscovery.com/
Acknowledgements
The authors would like to thank all of the Cell Line Engineering team at Horizon Discovery Ltd for their work in developing and optimizing these protocols. We are also grateful to Prof. David Russell (University of Washington) and Prof. Eric Hendrickson (University of Minnesota) for their advice and constructive discussions during the optimization process. We would also like to thank Eric Rhodes for his helpful comments during writing of the manuscript.
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Howes, R., Schofield, C. (2015). Genome Engineering Using Adeno-Associated Virus (AAV). In: Pruett-Miller, S. (eds) Chromosomal Mutagenesis. Methods in Molecular Biology, vol 1239. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-1862-1_5
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DOI: https://doi.org/10.1007/978-1-4939-1862-1_5
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