Efficient ΦC31 integrase–mediated site-specific germline transformation of Anopheles gambiae

Pondeville, Emilie; Puchot, Nicolas; Meredith, Janet; Lynd, Amy; Vernick, Kenneth D.; Lycett, Gareth; Eggleston, Paul; Bourgouin, Catherine

doi:10.1038/nprot.2014.117

Cited by 45 publications

(31 citation statements)

References 47 publications

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“…Larvae were raised in deionized water and fed finely ground TetraMin fish food. Embryo microinjection was performed essentially as described ( Fuchs et al 2013 ; Pondeville et al 2014 ). Freshly laid eggs were directly aligned against the edge of a nitrocellulose membrane kept wet with overlaying filter paper soaked with demineralized water.…”

Section: Methodsmentioning

confidence: 99%

Tools forAnopheles gambiaeTransgenesis

Volohonsky

Terenzi

Soichot

et al. 2015

G3 Genes|Genomes|Genetics

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Transgenesis is an essential tool to investigate gene function and to introduce desired characters in laboratory organisms. Setting-up transgenesis in non-model organisms is challenging due to the diversity of biological life traits and due to knowledge gaps in genomic information. Some procedures will be broadly applicable to many organisms, and others have to be specifically developed for the target species. Transgenesis in disease vector mosquitoes has existed since the 2000s but has remained limited by the delicate biology of these insects. Here, we report a compilation of the transgenesis tools that we have designed for the malaria vector Anopheles gambiae, including new docking strains, convenient transgenesis plasmids, a puromycin resistance selection marker, mosquitoes expressing cre recombinase, and various reporter lines defining the activity of cloned promoters. This toolbox contributed to rendering transgenesis routine in this species and is now enabling the development of increasingly refined genetic manipulations such as targeted mutagenesis. Some of the reagents and procedures reported here are easily transferable to other nonmodel species, including other disease vector or agricultural pest insects.

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Section: Methodsmentioning

confidence: 99%

Tools forAnopheles gambiaeTransgenesis

Volohonsky

Terenzi

Soichot

et al. 2015

G3 Genes|Genomes|Genetics

110

150

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“…Screening was performed by standard methods (Lobo et al, 2006;Pondeville et al, 2014;Li et al, 2017bLi et al, , 2018. Briefly, the phenotype of G 0 and G 1 mosquitoes was assessed and photographed under a Leica Biosystems (Nussloch, Germany) M165 FC stereomicroscope.…”

Section: Mutation Screensmentioning

confidence: 99%

Methods for the generation of heritable germline mutations in the disease vector Culex quinquefasciatus using clustered regularly interspaced short palindrome repeats‐associated protein 9

Liu

et al. 2019

Insect Molecular Biology

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Culex quinquefasciatus is a vector of many diseases that adversely impact human and animal health; however, compared to other mosquito vectors limited genome engineering technologies have been characterized for this vector. Clustered regularly interspaced short palindrome repeats‐associated protein 9 (CRISPR‐Cas9) based technologies are a powerful tool for genome engineering and functional genetics and consequently have transformed genetic studies in many organisms. Our objective was to improve upon the limited technologies available for genome editing in C. quinquefasciatus to create a reproducible and straightforward method for CRISPR‐Cas9‐targeted mutagenesis in this vector. Here we describe methods to achieve high embryo survival and mutagenesis rates and we provide details on the injection supplies and procedures, embryo handling and guide RNA (gRNA) target designs. Through these efforts, we achieved embryo survival rates and germline mutagenesis rates that greatly exceed previously reported rates in this vector. This work is also the first to characterize the white gene marker in this species, which is a valuable phenotypic marker for future transgenesis or mutagenesis of this vector. Overall, these tools provide the framework for future functional genetic studies in this important disease vector and may support the development of future gene drive and genetic technologies that can be used to control this vector.

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“…Considering half of G0 individuals were sterile, the transformation efficiency is estimated ranging from 2.8% (if only one out of the four G0 males produced transgenic gametes) to 7% (if the four G0 males produced transgenic gametes). Preparation of the injection mix, microinjections, screening and stable homozygous line generation were carried out as previously described (Pondeville, Puchot et al 2014). Correct site-specific integration was confirmed by PCR across the resulting attL and attR using specific primers (Meredith, Basu et al 2011).…”

Section: Hml-gal4 Line Establishmentmentioning

confidence: 99%

Hemocyte-targeted gene expression in the female malaria mosquito using thehemolectinpromoter fromDrosophila

Pondeville

Puchot

Parvy

et al. 2019

Preprint

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Hemocytes, the immune cells in mosquitoes, participate in immune defenses against pathogens including malaria parasites. Mosquito hemocytes can also be infected by arthropod-borne viruses but the pro-or anti-viral nature of this interaction is unknown.Although there has been progress on hemocyte characterization during pathogen infections in mosquitoes, the specific contribution of hemocytes to immune responses and the hemocytespecific functions of immune genes and pathways remain unresolved due to the lack of genetic tools to manipulate gene expression in these cells specifically. Here, we used the Gal4-UAS system to characterize the activity of the Drosophila hemocyte-specific hemolectin promoter in the adults of Anopheles gambiae, the malaria mosquito. We established an hml-Gal4 driver line that we further crossed to a fluorescent UAS responder line, and examined the expression pattern in the adult progeny driven by the hml promoter. We show that the hml regulatory region drives hemocyte-specific transgene expression in a subset of hemocytes, and that transgene expression is triggered after a blood meal. The hml promoter drives transgene expression in differentiating prohemocytes as well as in differentiated granulocytes.Analysis of different immune markers in hemocytes in which the hml promoter drives transgene expression revealed that this regulatory region could be used to study phagocytosis as well as melanization. Finally, the hml promoter drives transgene expression in hemocytes in which o'nyong'nyong virus replicates. Altogether, the hml promoter constitutes a good tool to drive transgene expression in hemocyte only and to analyze the function of these cells and the genes they express during pathogen infection in Anopheles gambiae.

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Efficient ΦC31 integrase–mediated site-specific germline transformation of Anopheles gambiae

Cited by 45 publications

References 47 publications

Tools forAnopheles gambiaeTransgenesis

Tools forAnopheles gambiaeTransgenesis

Methods for the generation of heritable germline mutations in the disease vector Culex quinquefasciatus using clustered regularly interspaced short palindrome repeats‐associated protein 9

Hemocyte-targeted gene expression in the female malaria mosquito using thehemolectinpromoter fromDrosophila

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