ASIS-Seq: Transgene Insertion Site Mapping by Nanopore Adaptive Sampling.
Methods Mol Biol
; 2631: 135-153, 2023.
Article
em En
| MEDLINE
| ID: mdl-36995666
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 91481-1486, 2019) or have limitations (Goodwin et al., Genome Research 29494-505, 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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Texto completo:
1
Base de dados:
MEDLINE
Assunto principal:
Nanoporos
Limite:
Animals
Idioma:
En
Revista:
Methods Mol Biol
Assunto da revista:
BIOLOGIA MOLECULAR
Ano de publicação:
2023
Tipo de documento:
Article
País de afiliação:
Estados Unidos