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Generating Transgenic Plants with Single-copy Insertions Using BIBAC-GW Binary Vector

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Here and there: the double-side transgene localization.

P A Salnikov¹, A A Khabarova¹, G S Koksharova¹

¹Institute of Cytology and Genetics of the Siberian Branch of the Russian Academy of Sciences, Novosibirsk, Russia.

Vavilovskii Zhurnal Genetiki I Selektsii

|November 10, 2021

Summary

This summary is machine-generated.

We developed a novel, cost-effective next-generation sequencing (NGS) method for precisely mapping both flanking regions of transposon insertions. This technique enhances genome-wide screening and chromosome engineering applications.

Keywords:

genome-wide screening sleeping beauty transposon transgene mapping transgenesis

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Area of Science:

Genomics
Molecular Biology
Bioinformatics

Background:

Random transgene integration is crucial for genome-wide screening, functional gene annotation, and regulatory element identification.
Existing transgene mapping methods like Inverse-PCR and LAM-PCR struggle to simultaneously localize both flanking regions of a transgene.
Precise localization and artifact filtration are critical for the accuracy of these screening techniques.

Purpose of the Study:

To develop a cost-effective and simple next-generation sequencing (NGS)-based approach for simultaneously mapping both flanking regions of transgene insertions.
To overcome the limitations of existing methods that cannot provide simultaneous localization of both transgene flanking regions.
To enable precise insertion mapping and facilitate screening approaches in chromosome engineering.

Main Methods:

Designed vectors lacking specific restriction enzyme recognition sites to facilitate DNA fragmentation.
Utilized DNA looping and sequencing of fragmented DNA to generate data linking both flanking regions of the transposon.
Applied the method for mapping Sleeping Beauty (SB) transposon integration in the human HAP1 cell line.

Main Results:

Successfully developed and demonstrated a cheap and simple NGS-based assay for precise transgene integration mapping.
The assay efficiently localized genomic transposon integrations, with results confirmed by PCR analysis.
The method enables the linking of both flanking regions of a transposon, overcoming previous technical limitations.

Conclusions:

The developed NGS approach provides a powerful tool for precise transgene insertion mapping, overcoming limitations of existing methods.
This technique is valuable for applications requiring simultaneous localization of both transgene flanking regions, such as chromosome engineering and detecting rearrangements.
The method offers a practical solution for multiplex transgene localization and analysis of inter-transgene rearrangements.