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Objective <p>DNA repair pathways have been leveraged for genetic engineering strategies, including self-eliminating transgenes in insects. In effort towards establishing a self-eliminating transgenic system that leaves desired cargoes like antibodies or anti-viral molecules in <i>Aedes aegypti</i>, steps were taken to generate a plasmid with direct repeats and other necessary transgenic components. Here, we present an account of series of recombination events in <i>E. coli</i> that were encountered during the building of a multi-component plasmid for achieving a cargo-leaving self-eliminating transgenic system in <i>Ae. aegypti</i>.</p> Results <p>Step-wise construction of the multi-component plasmid in Stellar™ <i>E. coli</i> competent cells achieved successful modifications of a parent plasmid from a previous study by removing some DNA fragments, incorporating direct repeats and incorporating fluorescent marker gene cassettes (3xP3-DsRed and 3xP3-BFP) in desired locations. However, recombination events resulting in precise and repeatable deletions of small and large DNA fragments were obtained following incorporation of a target site for the <i>in-cis</i>-located <i>I-SceI</i> homing endonuclease into the plasmid. These events were also observed using other <i>E. coli</i> strains like SURE and NEB<sup><i>®</i></sup> Stable competent cells, although one clone from the NEB<sup>®</sup> Stable cells eventually yielded the expected plasmid construct. Analysis of the incorrect plasmids revealed that SSA-like and MMEJ-like DNA repair pathways appeared responsible for the recombination events encountered, suggesting that <i>Aedes aegypti</i> regulatory elements used for control of the homing endonuclease might be leaky in <i>E. coli</i>.</p>

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SSA-like and MMEJ-like recombination events in a transgenic construct with in-cis programmed self-eliminating components in different E. coli strains

  • 期刊论文

  • 出版模式：

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  • 发表日期：

    2026年2月6日

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  • Christian E. Ogaugwu,
  • Zach N. Adelman

摘要

Objective

DNA repair pathways have been leveraged for genetic engineering strategies, including self-eliminating transgenes in insects. In effort towards establishing a self-eliminating transgenic system that leaves desired cargoes like antibodies or anti-viral molecules in Aedes aegypti, steps were taken to generate a plasmid with direct repeats and other necessary transgenic components. Here, we present an account of series of recombination events in E. coli that were encountered during the building of a multi-component plasmid for achieving a cargo-leaving self-eliminating transgenic system in Ae. aegypti.

Results

Step-wise construction of the multi-component plasmid in Stellar™ E. coli competent cells achieved successful modifications of a parent plasmid from a previous study by removing some DNA fragments, incorporating direct repeats and incorporating fluorescent marker gene cassettes (3xP3-DsRed and 3xP3-BFP) in desired locations. However, recombination events resulting in precise and repeatable deletions of small and large DNA fragments were obtained following incorporation of a target site for the in-cis-located I-SceI homing endonuclease into the plasmid. These events were also observed using other E. coli strains like SURE and NEB® Stable competent cells, although one clone from the NEB® Stable cells eventually yielded the expected plasmid construct. Analysis of the incorrect plasmids revealed that SSA-like and MMEJ-like DNA repair pathways appeared responsible for the recombination events encountered, suggesting that Aedes aegypti regulatory elements used for control of the homing endonuclease might be leaky in E. coli.