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Recombinant DNA technology called transgenesis is often used to add a foreign gene or remove a detrimental gene from an organism. Such genetically modified organisms are called transgenic organisms.
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A modular gene targeting system for sequential transgene stacking in plants.

Sandeep Kumar1, Diaa AlAbed1, Andrew Worden1

  • 1Dow AgroSciences LLC, 9330 Zionsville Road, Indianapolis, IN 46268, USA.

Journal of Biotechnology
|April 28, 2015
PubMed
Summary
This summary is machine-generated.

This study introduces a novel gene targeting method for creating multigene stacks in maize. The system achieves high-frequency, site-specific transgene integration using zinc finger nucleases and a unique selectable marker strategy.

Keywords:
CornGene targetingPlantsTransgene stackingZinc finger nuclease

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Area of Science:

  • Molecular Biology
  • Plant Biotechnology
  • Genetics

Background:

  • Creating multigene stacks for complex trait engineering requires efficient site-specific transgene integration.
  • Previous gene targeting methods often rely on partial or defective selectable markers, limiting efficiency and recyclability.

Purpose of the Study:

  • To develop a modular, selection-based method for site-specific integration of transgenes into a genomic locus.
  • To create multigene stacks with high-frequency gene targeting and improved selectable marker recycling.

Main Methods:

  • Utilized zinc finger nucleases (ZFNs) to induce double-strand breaks (DSBs) at a pre-defined genomic locus.
  • Employed a unique intron downstream of a promoter driving a selectable marker gene to facilitate homology and selection.
  • Tested the system in maize using the pat selectable marker gene on Bialaphos-containing medium.

Main Results:

  • Achieved high-frequency gene targeting with up to 30% of regenerated maize plants showing successful donor construct integration.
  • The selection system effectively reduced regeneration from random insertions of promoterless donor constructs.
  • Demonstrated the potential for exchanging functional selectable marker genes in successive gene targeting cycles.

Conclusions:

  • The developed method enables efficient, site-specific creation of multigene stacks through modular gene targeting.
  • The system allows for the recycling of functional selectable marker genes, facilitating multiple generations of gene targeting.
  • This approach offers a robust strategy for complex genetic engineering in plants like maize.