RNAi in the Malaria Vector, Anopheles gambiae

Catteruccia, Flaminia; Levashina, Elena A.

doi:10.1007/978-1-60327-295-7_5

Cited by 21 publications

(19 citation statements)

References 9 publications

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“…Current methods for inducing non-transgenic RNAi in mosquitoes involve direct injection of dsRNA into the adult hemocoel 12,13 or larval feeding of RNAi trigger-coated nanoparticles [14][15][16][17] or microalgae-based dsRNA molecules 18 . Targeting the adult mosquito, while extremely valuable, can exclude a large number of genes that function during earlier developmental periods.…”

Section: Discussionmentioning

confidence: 99%

RNAi Trigger Delivery into <em>Anopheles gambiae</em> Pupae

Regna

Harrison

Heyse

et al. 2016

JoVE

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RNA interference (RNAi), a naturally occurring phenomenon in eukaryotic organisms, is an extremely valuable tool that can be utilized in the laboratory for functional genomic studies. The ability to knockdown individual genes selectively via this reverse genetic technique has allowed many researchers to rapidly uncover the biological roles of numerous genes within many organisms, by evaluation of loss-of-function phenotypes. In the major human malaria vector Anopheles gambiae, the predominant method used to reduce the function of targeted genes involves injection of double-stranded (dsRNA) into the hemocoel of the adult mosquito. While this method has been successful, gene knockdown in adults excludes the functional assessment of genes that are expressed and potentially play roles during pre-adult stages, as well as genes that are expressed in limited numbers of cells in adult mosquitoes. We describe a method for the injection of Serine Protease Inhibitor 2 (SRPN2) dsRNA during the early pupal stage and validate SRPN2 protein knockdown by observing decreased target protein levels and the formation of melanotic pseudo-tumors in SRPN2 knockdown adult mosquitoes. This evident phenotype has been described previously for adult stage knockdown of SRPN2 function, and we have recapitulated this adult phenotype by SRPN2 knockdown initiated during pupal development. When used in conjunction with a dye-labeled dsRNA solution, this technique enables easy visualization by simple light microscopy of injection quality and distribution of dsRNA in the hemocoel.

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Section: Discussionmentioning

confidence: 99%

RNAi Trigger Delivery into <em>Anopheles gambiae</em> Pupae

Regna

Harrison

Heyse

et al. 2016

JoVE

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“…Functional characterisation of these genes through transient RNAi is possible in adult An. gambiae in some cases [9], but this approach is limited, not least by the non-systemic nature of gene silencing in mosquitoes [10]. In addition, transgenic technology has been developed in this species [11], [12], [13], [14], [15], but has yet to be exploited extensively to analyse gene function through temporal and spatial mis-expression.…”

Section: Introductionmentioning

confidence: 99%

Development of the Bi-Partite Gal4-UAS System in the African Malaria Mosquito, Anopheles gambiae

Lynd

Lycett

2012

PLoS ONE

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Functional genetic analysis in Anopheles gambiae would be greatly improved by the development of a binary expression system, which would allow the more rapid and flexible characterisation of genes influencing disease transmission, including those involved in insecticide resistance, parasite interaction, host and mate seeking behaviour. The Gal4-UAS system, widely used in Drosophila melanogaster functional genetics, has been significantly modified to achieve robust application in several different species. Towards this end, previous work generated a series of modified Gal4 constructs that were up to 20 fold more active than the native gene in An. gambiae cells. To examine the Gal4-UAS system in vivo, transgenic An. gambiae driver lines carrying a modified Gal4 gene under the control of the carboxypeptidase promoter, and responder lines carrying UAS regulated luciferase and eYFP reporter genes have been created. Crossing of the Gal4 and UAS lines resulted in progeny that expressed both reporters in the expected midgut specific pattern. Although there was minor variation in reporter gene activity between the different crosses examined, the tissue specific expression pattern was consistent regardless of the genomic location of the transgene cassettes. The results show that the modified Gal4-UAS system can be used to successfully activate expression of transgenes in a robust and tissue specific manner in Anopheles gambiae. The midgut driver and dual reporter responder constructs are the first to be developed and tested successfully in transgenic An. gambiae and provide the basis for further advancement of the system in this and other insect species.

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“…This technique was generalized for the functional characterization of hundreds of mosquito genes [34]. However, injection per se was reported to impinge on Plasmodium development by the potential induction of the wounding response [35].…”

Section: Resultsmentioning

confidence: 99%

Targeted Mutagenesis in the Malaria Mosquito Using TALE Nucleases

et al. 2013

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Anopheles gambiae, the main mosquito vector of human malaria, is a challenging organism to manipulate genetically. As a consequence, reverse genetics studies in this disease vector have been largely limited to RNA interference experiments. Here, we report the targeted disruption of the immunity gene TEP1 using transgenic expression of Transcription-Activator Like Effector Nucleases (TALENs), and the isolation of several TEP1 mutant A. gambiae lines. These mutations inhibited protein production and rendered TEP1 mutants hypersusceptible to Plasmodium berghei. The TALEN technology opens up new avenues for genetic analysis in this disease vector and may offer novel biotechnology-based approaches for malaria control.

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RNAi in the Malaria Vector, Anopheles gambiae

Cited by 21 publications

References 9 publications

RNAi Trigger Delivery into <em>Anopheles gambiae</em> Pupae

RNAi Trigger Delivery into <em>Anopheles gambiae</em> Pupae

Development of the Bi-Partite Gal4-UAS System in the African Malaria Mosquito, Anopheles gambiae

Targeted Mutagenesis in the Malaria Mosquito Using TALE Nucleases

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