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

Area of Science:

• Genomics
• Molecular Biology
• Biotechnology

Background:

• Transgenic mice are crucial for biomedical research, aiding studies in gene expression, developmental biology, and disease modeling.
• Random DNA integration in transgenic models can cause insertional mutagenesis and transgene silencing, complicating research.
• Current methods for identifying transgene integration sites are often laborious or limited in scope.

Purpose of the Study:

• To present a novel, efficient method for accurately locating transgene integration sites in the host genome.
• To overcome the limitations of existing burdensome or restricted techniques for transgene insertion site mapping.
• To facilitate the characterization of transgenic lines for improved biomedical research.

Main Methods:

• Developed Adaptive Sampling Insertion Site Sequencing (ASIS-Seq), a targeted sequencing approach.
• Utilized Oxford Nanopore Technologies (ONT) sequencers for transgene integration site identification.
• Optimized the method for minimal DNA input (3 µg) and streamlined sample preparation (3 hours).

Main Results:

• ASIS-Seq effectively locates transgene integration sites within the host genome.
• The method requires significantly less hands-on time and sequencing duration compared to traditional approaches.
• Successful application demonstrated for identifying transgene locations in mouse models.

Conclusions:

• ASIS-Seq offers a robust and accessible solution for mapping transgene insertion sites.
• This technique enhances the utility of transgenic mouse models by enabling precise genomic characterization.
• The method supports advancements in gene expression studies, developmental biology, and the creation of genetic disease models and therapies.