Molecular Genetic Manipulation of Vector Mosquitoes

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The discovery and mapping of cis-regulatory elements is important for understanding regulation of gene transcription in mosquito vectors of human diseases. Genome sequence data are available for 3 species, Aedes aegypti, Anopheles gambiae, and Culex quinquefasciatus (Diptera: Culicidae), representing 2 subfamilies (Culicinae and Anophelinae) that are estimated to have diverged 145 to 200 million years ago. Comparative genomics tools were used to screen genomic DNA fragments located in the 5 -end flanking regions of orthologous genes. These analyses resulted in the identification of 137 sequences, designated ''mosquito motifs,'' 7 to 9 nucleotides in length, representing 18 families of putative cis-regulatory elements conserved significantly among the 3 species when compared to the fruit fly, Drosophila melanogaster. Forty-one of the motifs were implicated previously in experiments as sites for binding transcription factors or functioning in the regulation of mosquito gene expression. Further analyses revealed associations between specific motifs and expression profiles, particularly in those genes that show increased or decreased mRNA abundance in females following a blood meal, and those accumulating transcription products exclusively or preferentially in the midgut, fat bodies, or ovaries. These results validate the methodology and support a relationship between the discovered motifs and the conservation of hematophagy in mosquitoes.gene expression ͉ hematophagy ͉ Aedes ͉ Anopheles ͉ Culex

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confidence: 99%

Comparative genomics allows the discovery of cis -regulatory elements in mosquitoes

Sieglaff

Dunn

Xie

et al. 2009

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“…The ability to develop new vector-control approaches depends in part on continued growth in the understanding of vector biology. The significant expansion of knowledge of Anopheles mosquitoes has been stimulated by the availability of new resources (e.g., whole genome sequences) and technologies (e.g., transgenics) (6,7). "Reverse-genetics" approaches are being used in contemporary functional genomics studies, whereby "mutant" phenotypes are created to infer gene function.…”

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confidence: 99%

piggyBac transposon remobilization and enhancer detection in Anopheles mosquitoes

O’Brochta

Alford²,

Pilitt³

et al. 2011

Technical advances in mosquito biology are enabling the development of new approaches to vector control. Absent are powerful forward-genetics technologies, such as enhancer and gene traps, that permit determination of gene functions from the phenotypes arising from transposon insertion mutations. We show that the piggyBac transposon is highly active in the germline of the human malaria vector Anopheles stephensi. Up to 6% of the progeny from transgenic A. stephensi containing a single 6-kb piggyBac element with a marker gene expressing EGFP had the vector in new genomic locations when piggyBac transposase was provided in trans from a second integrated transgene. The active transposition of piggyBac resulted in the efficient detection of enhancers, with ∼10% of the progeny with piggyBac in new locations with novel patterns of EGFP expression in third and fourth instar larvae and in adults. The availability of advanced transgenic capabilities such as efficient transposon-based forward-genetics technologies for Anopheles mosquitoes not only will accelerate our understanding of mosquito functional genomics and the development of novel vector and disease transmission control strategies, but also will enable studies by evolutionary developmental biologists, virologists, and parasitologists.transposable element | Plasmodium | Drosophila

“…Insecticide and parasite drug resistance continue to hinder malaria eradication efforts and innovative approaches to disease control are needed to complement traditional methods (2). One novel approach proposes the use of transgenic mosquitoes to introgress a parasite resistance gene into wild, malaria-susceptible mosquito populations, thereby interrupting transmission (3)(4)(5).…”

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confidence: 99%

Transgenic Anopheles stephensi coexpressing single-chain antibodies resist Plasmodium falciparum development

Isaacs

Jasinskiene

Третьяков

et al. 2012

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129

150

Anopheles stephensi mosquitoes expressing m1C3, m4B7, or m2A10 single-chain antibodies (scFvs) have significantly lower levels of infection compared to controls when challenged with Plasmodium falciparum, a human malaria pathogen. These scFvs are derived from antibodies specific to a parasite chitinase, the 25 kDa protein and the circumsporozoite protein, respectively. Transgenes comprising m2A10 in combination with either m1C3 or m4B7 were inserted into previously-characterized mosquito chromosomal "docking" sites using site-specific recombination. Transgene expression was evaluated at four different genomic locations and a docking site that permitted tissue-and sex-specific expression was researched further. Fitness studies of docking site and dual scFv transgene strains detected only one significant fitness cost: adult docking-site males displayed a late-onset reduction in survival. The m4B7/m2A10 mosquitoes challenged with P. falciparum had few or no sporozoites, the parasite stage infective to humans, in three of four experiments. No sporozoites were detected in m1C3/m2A10 mosquitoes in challenge experiments when both genes were induced at developmentally relevant times. These studies support the conclusion that expression of a single copy of a dual scFv transgene can completely inhibit parasite development without imposing a fitness cost on the mosquito.mosquito vector | population replacement | genetic engineering