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ASIS-Seq: Transgene Insertion Site Mapping by Nanopore Adaptive Sampling

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Transgenesis

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2631))

Abstract

Generation of transgenic mice by direct microinjection of foreign DNA into fertilized ova has become a routine technique in biomedical research. It remains an essential tool for studying gene expression, developmental biology, genetic disease models, and their therapies. However, the random integration of foreign DNA into the host genome that is inherent to this technology can lead to confounding effects associated with insertional mutagenesis and transgene silencing. Locations of most transgenic lines remain unknown because the methods are often burdensome (Nicholls et al., G3: Genes Genomes Genetics 9:1481–1486, 2019) or have limitations (Goodwin et al., Genome Research 29:494–505, 2019). Here, we present a method that we call Adaptive Sampling Insertion Site Sequencing (ASIS-Seq) to locate transgene integration sites using targeted sequencing on Oxford Nanopore Technologies’ (ONT) sequencers. ASIS-Seq requires only about 3 ug of genomic DNA, 3 hours of hands-on sample preparation time, and 3 days of sequencing time to locate transgenes in a host genome.

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Author information

Authors and Affiliations

  1. Genentech, Inc., Department of Molecular Biology, South San Francisco, CA, USA

    Charles Yu, Roger Caothien, Anna Pham, Lucinda Tam, Tuija Alcantar, Natasha Bacarro, Juan Reyes Jr, Marques Jackson, Brian Nakao & Merone Roose-Girma

Authors
  1. Charles Yu
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  2. Roger Caothien
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  3. Anna Pham
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  4. Lucinda Tam
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  5. Tuija Alcantar
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  6. Natasha Bacarro
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  7. Juan Reyes Jr
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  8. Marques Jackson
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  9. Brian Nakao
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  10. Merone Roose-Girma
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Corresponding author

Correspondence to Merone Roose-Girma .

Editor information

Editors and Affiliations

  1. Transgenic Animal Model Core, University of Michigan, Ann Arbor, MI, USA

    Thomas L. Saunders

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© 2023 The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature

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Yu, C. et al. (2023). ASIS-Seq: Transgene Insertion Site Mapping by Nanopore Adaptive Sampling. In: Saunders, T.L. (eds) Transgenesis. Methods in Molecular Biology, vol 2631. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-2990-1_5

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  • DOI: https://doi.org/10.1007/978-1-0716-2990-1_5

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-2989-5

  • Online ISBN: 978-1-0716-2990-1

  • eBook Packages: Springer Protocols

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