Using the GAL4-UAS System for Functional Genetics in &lt;em&gt;Anopheles gambiae&lt;/em&gt;

Poulton, Beth C; Colman, Fraser; Anthousi, Amalia; Grigoraki, Linda; Adolfi, Adriana; Lynd, Amy; Lycett, Gareth

doi:10.3791/62131

Cited by 7 publications

(6 citation statements)

References 0 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Genetic modification of An. gambiae has demonstrated that disruption of the ecdysone pathway is associated with embryonic lethality midway through development (Poulton et al 2021). Furthermore, a recent study of mosquito larvae demonstrated that larvicidal activity was correlated with inhibition of Nobo (Inaba et al 2022).…”

Section: Discussionmentioning

confidence: 99%

Potent GST ketosteroid isomerase activity relevant to ecdysteroidogenesis in the malaria vectorAnopheles gambiae

Musdal

Ismail

Sjödin

et al. 2023

Preprint

View full text Add to dashboard Cite

Nobo is a glutathione transferase (GST) crucially contributing to ecdysteroid biosynthesis in insects of the ordersDipteraandLepidoptera. Ecdysone is a vital steroid hormone in insects, which governs larval molting and metamorphosis, and suppression of its synthesis has potential as a novel approach to insect growth regulation and combatting vectors of disease. In general, GSTs catalyze detoxication, whereas the specific function of Nobo in ecdysteroidogenesis is unknown. We report that Nobo from the malaria-spreading mosquitoAnopheles gambiaeis a highly efficient ketosteroid isomerase catalyzing double-bond isomerization in the steroids 5-androsten-3,17-dione and 5-pregnen-3,20-dione. These mammalian ketosteroids are unknown in mosquitoes, but the discovered prominent catalytic activity with these compounds suggests that the unknown Nobo substrate in insects has a ketosteroid functionality. Nobo Asp111 is essential for activity with the steroids, but not for conventional GST substrates. Further characterization of Nobo may guide the development of new insecticides to prevent malaria.

show abstract

Section: Discussionmentioning

confidence: 99%

Potent GST ketosteroid isomerase activity relevant to ecdysteroidogenesis in the malaria vectorAnopheles gambiae

Musdal

Ismail

Sjödin

et al. 2023

Preprint

View full text Add to dashboard Cite

show abstract

“…The Drosophila binary GAL4-UAS system is famous for temporal and tissuespecific regulation of transgene DNA expression with continuing innovation in this genetic technology [37,50,53]. Some groups have ported the binary GAL4-UAS system to Aedes and Anopheles mosquito strains [54][55][56][57], but the pace of deploying these transgenic systems broadly to the rest of the mosquito research community has not been rapid. Rather, a new binary transgenic platform called the Q-system from the Potter lab was first developed in Drosophila to be orthogonal to the GAL4-UAS system and [58,59] has now been successfully applied to An.…”

Section: Why Think Of Fruit Flies When Mosquitoes Are Humanity's Bigg...mentioning

confidence: 99%

Transposon and Transgene Tribulations in Mosquitoes: A Perspective of piRNA Proportions

Lau,

Macias

2024

DNA

View full text Add to dashboard Cite

Mosquitoes, like Drosophila, are dipterans, the order of “true flies” characterized by a single set of two wings. Drosophila are prime model organisms for biomedical research, while mosquito researchers struggle to establish robust molecular biology in these that are arguably the most dangerous vectors of human pathogens. Both insects utilize the RNA interference (RNAi) pathway to generate small RNAs to silence transposons and viruses, yet details are emerging that several RNAi features are unique to each insect family, such as how culicine mosquitoes have evolved extreme genomic feature differences connected to their unique RNAi features. A major technical difference in the molecular genetic studies of these insects is that generating stable transgenic animals are routine in Drosophila but still variable in stability in mosquitoes, despite genomic DNA-editing advances. By comparing and contrasting the differences in the RNAi pathways of Drosophila and mosquitoes, in this review we propose a hypothesis that transgene DNAs are possibly more intensely targeted by mosquito RNAi pathways and chromatin regulatory pathways than in Drosophila. We review the latest findings on mosquito RNAi pathways, which are still much less well understood than in Drosophila, and we speculate that deeper study into how mosquitoes modulate transposons and viruses with Piwi-interacting RNAs (piRNAs) will yield clues to improving transgene DNA expression stability in transgenic mosquitoes.

show abstract

“…To generate the UAS-CCEae3A responder plasmid (Fig 1B ), the CCEae3A cDNA (AAEL023844-RA) was first subcloned into a UAS plasmid (pSL*attB:YFP:Gyp:UAS14i:Gyp: attB) [10] previously described in [13]. The 1669-bp CCEae3A cDNA sequence was amplified from a template derived from the temephos resistant Nakhon Sawan 2 Ae.…”

Section: Gal4/uas and Uas:cceae3a Donor Plasmid Constructionmentioning

confidence: 99%

“…Finally, in vivo validation is sought through heterologous expression of candidates. and subsequent characterisation of insecticide resistance phenotypes [10][11][12][13]. To examine insecticide resistance in mosquitoes, we have adapted the widely used GAL4-UAS and phiC31 systems to perform functional analysis of the most promising cytochrome mosquito P450, GST and sensory appendage protein (SAP) candidates in An.…”

Section: Introductionmentioning

confidence: 99%

Aedes aegypti CCEae3A carboxylase expression confers carbamate, organophosphate and limited pyrethroid resistance in a model transgenic mosquito

Poulton,

Colman,

Anthousi

et al. 2024

PLoS Negl Trop Dis

View full text Add to dashboard Cite

Insecticide resistance is a serious threat to our ability to control mosquito vectors which transmit pathogens including malaria parasites and arboviruses. Understanding the underlying mechanisms is an essential first step in tackling the challenges presented by resistance. This study aimed to functionally characterise the carboxylesterase, CCEae3A, the elevated expression of which has been implicated in temephos resistance in Aedes aegypti and Aedes albopictus larvae. Using our GAL4/UAS expression system, already established in insecticide-sensitive Anopheles gambiae mosquitoes, we produced transgenic An. gambiae mosquitoes that express an Ae. aegypti CCEae3A ubiquitously. This new transgenic line permits examination of CCEae3A expression in a background in which there is not a clear orthologue in Vectorbase and allows comparison with existing An. gambiae GAL4-UAS lines. Insecticide resistance profiling of these transgenic An. gambiae larvae indicated significant increases in resistance ratio for three organophosphate insecticides, temephos (6), chloropyriphos (6.6) and fenthion (3.2) when compared to the parental strain. Cross resistance to adulticides from three major insecticide classes: organophosphates (malathion, fenitrothion and pirimiphos methyl), carbamates (bendiocarb and propoxur) and pyrethroid (alpha-cypermethrin) was also detected. Resistance to certain organophosphates and carbamates validates conclusions drawn from previous expression and phenotypic data. However, detection of resistance to pirimiphos methyl and alphacypermethrin has not previously been formally associated with CCEae3A, despite occurring in Ae. aegypti strains where this gene was upregulated. Our findings highlight the importance of characterising individual resistance mechanisms, thereby ensuring accurate information is used to guide future vector control strategies.

show abstract

Using the GAL4-UAS System for Functional Genetics in <em>Anopheles gambiae</em>

Cited by 7 publications

References 0 publications

Potent GST ketosteroid isomerase activity relevant to ecdysteroidogenesis in the malaria vectorAnopheles gambiae

Potent GST ketosteroid isomerase activity relevant to ecdysteroidogenesis in the malaria vectorAnopheles gambiae

Transposon and Transgene Tribulations in Mosquitoes: A Perspective of piRNA Proportions

Aedes aegypti CCEae3A carboxylase expression confers carbamate, organophosphate and limited pyrethroid resistance in a model transgenic mosquito

Contact Info

Product

Resources

About