Genetic transformation of mosquitoes by microparticle bombardment

Abstract

Mosquitoes constitute the major living beings causing human deaths in the world. They are vectors of malaria, yellow fever, dengue, zika, filariases, chikungunya, among other diseases. New strategies to control/eradicate mosquito populations are based on newly developed genetic manipulation techniques. However, genetic transformation of mosquitoes is a major technical bottleneck due to low efficiency, the need of sophisticated equipment, and highly trained personnel. The present report shows the transgenerational genetic transformation of Aedes aegypti, using the particle inflow gun (PIG), by integrating the ecfp gene in the AAEL000582 mosquito gene with the CRISPR‐Cas9 technique, achieving a mean efficiency of 44.5% of bombarded individuals (G0) that showed ECFP expression in their tissues, and a mean of 28.5% transformation efficiency measured on G1 individuals. The same transformation technique was used to integrate the egfp/scorpine genes cloned in the Minos transposon pMinHygeGFP into the Anopheles albimanus genome, achieving a mean efficiency of 43.25% of bombarded individuals (G0) that showed EGFP expression in their tissues. Once the technique was standardized, transformation of Ae. aegypti neonate larvae and An. albimanus eggs was achieved when exposed to gold microparticle bombardment. Integration of genes and heterologous protein expression were confirmed by PCR, sequencing, fluorescent microscopy, mass spectrometry, Western blot and dot blot analyses. Transgenerational inheritance of the transgenes was observed only on Ae. aegypti, as all transformed An. albimanus individuals died at the pupal stage of the G0 generation.

Differentiated impacts of human interventions on nature

“…It is this capability that "random"-or more accurately unguided-mutagenic processes do not have and why it can be difficult or effectively impossible to apply them to the same desired ends. The ability to rapidly change and return to a particular locus for serial changes, or to treat multiple different parts of a genome simultaneously, decreases the time required to engineer desired phenotypes into existence (Lim and Choi, 2019;Reis et al, 2019;Riesenberg et al, 2019;Wolter et al, 2019). At a remarkable frequency of one in only 67 treated protoplasts (>0.02%), CRISPR-Cas9 performed simultaneous changes in two different genes to generate a new color in petunia.…”

“…Furthermore, even in species and cultivars where plant transformation is possible, the process is expensive, slow, requires significant resources in terms of facilities and expertise, is frequently inefficient, and damages the plant genome. (Doyle et al, 2019) Various and equally vexing challenges of the type referred to by Doyle et al (2019) target organisms-bacteria, fungi, plants, and animalswith each requiring customized methods with many methods working for only some species of each group (e.g., Yoshida and Sato, 2009;Rivera et al, 2014;Kelliher et al, 2019;Ren et al, 2019;Lule-Chávez et al, 2020). The difficulty in developing and deploying customized methods and materials, along with highly specialized expertise, limits the ability of gene technology to amplify the risks.…”

“…An examination of these new products is well beyond the scope of this article but was recently reviewed by us (Heinemann and Walker, 2019). Being able to easily penetrate living tissue of all organisms with DNA, RNA, and proteins, even achieving this delivery at a lower efficiency than existing customized methods, adds a differential scalability to gene technologies (Lule-Chávez et al, 2020).…”