Targeted gene expression in the transgenic Aedes aegypti using the binary Gal4-UAS system

Kokoza, Vladimir; Raikhel, Alexander S.

doi:10.1016/j.ibmb.2011.04.004

Cited by 69 publications

(83 citation statements)

References 57 publications

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“…aegypti transgenic responder lines-M3-1, UAS-scramble-sponge; M3-2, UAS-miR-8-sponge-have been established previously (31). These responder lines were crossed with a Vg-Gal4 driver line, also developed before (66). Progeny was screened for concurrent presence of eye-specific selection markers, EGFP and dsRed.…”

Section: Methodsmentioning

confidence: 99%

Transcriptome-wide microRNA and target dynamics in the fat body during the gonadotrophic cycle of Aedes aegypti

Zhang

Aksoy

Girke

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

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The mosquito Aedes aegypti is a major vector of numerous viral diseases, because it requires a blood meal to facilitate egg development. The fat body, a counterpart of mammalian liver and adipose tissues, is the metabolic center, playing a key role in reproduction. Therefore, understanding of regulatory networks controlling its functions is critical, and the role of microRNAs (miRNAs) in the process is largely unknown. We aimed to explore miRNA expression and potential targets in the female fat body of Ae. aegypti, as well as their changes postblood meal (PBM). Small RNA library analysis revealed five unique miRNA patterns sequentially expressed at five sampled time points, likely responding to, and affecting, waves of upstream hormonal signals and gene expression in the same period. To link miRNA identities with downstream targets, transcriptome-wide mRNA 3′ UTR interaction sites were experimentally determined at 72 h posteclosion and 24 h PBM through Argonaute 1 cross-linking and immunoprecipitation followed by high-throughput sequencing. Several target sites were validated by means of in vitro luciferase assays with wild-type and mutated 3′ UTRs for six miRNA families. With established transgenic lines, consistent results were observed with spatiotemporal knockdown of miR-8 and luciferase assays. We further investigated miRNAs potentially regulating various physiological processes based on Clusters of Orthologous Groups functional categories. Hence, the present work comprehensively elucidated miRNA expression and target dynamics in the female mosquito fat body, providing a solid foundation for future functional studies of miRNA regulation during the gonadotrophic cycle.Aedes aegypti | fat body | microRNA | microRNA targets | Argonaute 1

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Section: Methodsmentioning

confidence: 99%

Transcriptome-wide microRNA and target dynamics in the fat body during the gonadotrophic cycle of Aedes aegypti

Zhang

Aksoy

Girke

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

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“…The Vg-Gal4 driver and pBac[3xP3-DsRed] transformation vectors were produced as previously described (13). The germline transformation vectors carrying the UAS-miR-8-SP and UAS-Scr-SP were constructed as described below.…”

Section: Methodsmentioning

confidence: 99%

“…The germline transformation for the Vg-Gal4 driver line was performed as described previously (13,29). PiggyBac-mediated germline transformation for the UAS-miR-8-SP and UAS-Scr-SP responder lines was carried out by the University of Maryland Institute for Bioscience and Biotechnology Research Insect Transformation Facility.…”

Section: Methodsmentioning

confidence: 99%

“…PiggyBac-mediated germline transformation for the UAS-miR-8-SP and UAS-Scr-SP responder lines was carried out by the University of Maryland Institute for Bioscience and Biotechnology Research Insect Transformation Facility. The Vg-Gal4/ UAS-miR-8-SP and Vg-Gal4/UAS-Scr-SP hybrid mosquito lines were established as previously described (13,29).…”

Section: Methodsmentioning

confidence: 99%

“…Because of the high conservation of miR-8, a Drosophila upstream activating sequence (UAS)-miR-8-SP cassette was used (12) in combination with the previously established Aedes Vitellogenin (Vg)-linked yeast transcriptional activator Gal4 protein/UAS system (13). The fat body-specific system uses the Vg gene promoter to drive transgene expression (13), permitting precise in vivo spatiotemporal genetic testing of miR-8 functions in the female mosquito fat body PBM (Fig. S3A).…”

Section: Mir-8 Is Enriched In the Female Mosquito Fat Body After Bloodmentioning

confidence: 99%

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MicroRNA-8 targets the Wingless signaling pathway in the female mosquito fat body to regulate reproductive processes

Lucas

Roy

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

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102

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Female mosquitoes require a blood meal for reproduction, and this blood meal provides the underlying mechanism for the spread of many important vector-borne diseases in humans. A deeper understanding of the molecular mechanisms linked to mosquito blood meal processes and reproductive events is of particular importance for devising innovative vector control strategies. We found that the conserved microRNA miR-8 is an essential regulator of mosquito reproductive events. Two strategies to inhibit miR-8 function in vivo were used for functional characterization: systemic antagomir depletion and spatiotemporal inhibition using the miRNA sponge transgenic method in combination with the yeast transcriptional activator gal4 protein/upstream activating sequence system. Depletion of miR-8 in the female mosquito results in defects related to egg development and deposition. We used a multialgorithm approach for miRNA target prediction in mosquito 3′ UTRs and experimentally verified secreted winglessinteracting molecule (swim) as an authentic target of miR-8. Our findings demonstrate that miR-8 controls the activity of the longrange Wingless (Wg) signaling by regulating Swim expression in the female fat body. We discovered that the miR-8/Wg axis is critical for the proper secretion of lipophorin and vitellogenin by the fat body and subsequent accumulation of these yolk protein precursors by developing oocytes.small RNA | microRNA | Wingless signaling | reproduction | mosquito

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Use of genetic markers in insect histology

2014

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Targeted gene expression in the transgenic Aedes aegypti using the binary Gal4-UAS system

Cited by 69 publications

References 57 publications

Transcriptome-wide microRNA and target dynamics in the fat body during the gonadotrophic cycle of Aedes aegypti

Transcriptome-wide microRNA and target dynamics in the fat body during the gonadotrophic cycle of Aedes aegypti

MicroRNA-8 targets the Wingless signaling pathway in the female mosquito fat body to regulate reproductive processes

Use of genetic markers in insect histology

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