Frontiers in Bioengineering and Biotechnology (Sep 2023)

Optimizing CRE and PhiC31 mediated recombination in Aedes aegypti

  • Leonela Z. Carabajal Paladino,
  • Ray Wilson,
  • Priscilla Y. L. Tng,
  • Vishaal Dhokiya,
  • Elizabeth Keen,
  • Piotr Cuber,
  • Will Larner,
  • Sara Rooney,
  • Melanie Nicholls,
  • Anastasia Uglow,
  • Luke Williams,
  • Michelle A. E. Anderson,
  • Sanjay Basu,
  • Philip T. Leftwich,
  • Luke Alphey

DOI
https://doi.org/10.3389/fbioe.2023.1254863
Journal volume & issue
Vol. 11

Abstract

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Introduction: Genetic manipulation of Aedes aegypti is key to developing a deeper understanding of this insects’ biology, vector-virus interactions and makes future genetic control strategies possible. Despite some advances, this process remains laborious and requires highly skilled researchers and specialist equipment.Methods: Here we present two improved methods for genetic manipulation in this species. Use of transgenic lines which express Cre recombinase and a plasmid-based method for expressing PhiC31 when injected into early embryos.Results: Use of transgenic lines which express Cre recombinase allowed, by simple crossing schemes, germline or somatic recombination of transgenes, which could be utilized for numerous genetic manipulations. PhiC31 integrase based methods for site-specific integration of genetic elements was also improved, by developing a plasmid which expresses PhiC31 when injected into early embryos, eliminating the need to use costly and unstable mRNA as is the current standard.Discussion: Here we have expanded the toolbox for synthetic biology in Ae. aegypti. These methods can be easily transferred into other mosquito and even insect species by identifying appropriate promoter sequences. This advances the ability to manipulate these insects for fundamental studies, and for more applied approaches for pest control.

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