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United States Department of Agriculture

Agricultural Research Service

Title: Flight Muscle-Specific Expression of Act88f:gfp in Transgenic Culex Quinquefusciatus Say (Diptera: Culicidae)

Authors
item Allen, Margaret
item Christensen, Bruce - UNIV WISCONSIN-MADISON

Submitted to: Parasitology International
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: April 1, 2004
Publication Date: December 1, 2004
Citation: Allen, M.L., Christensen, B.M. Flight muscle-specific expression of act88F:GFP in transgenic culex quinquefusciatus Say (Diptera: Culicidae). International Journal for Parasitology. Vol. 53/4, pp 307-314.

Interpretive Summary: Mosquitoes of the species Culex quinquefasciatus are important vectors of the parasitic nematodes that cause lymphatic filariasis in humans. About 120 milion people worldwide are afflicted. The parasitic nematodes require the mosquito not only for transportation to a human but to complete their life cycle. The nematodes reside in the flight muscles of susceptible mosquitoes where they mature to an infective stage that is then passed to humans when the mosquito takes a blood meal. But not all mosquitoes are susceptible to the nematode. Recent advances in genetic engineering have allowed us to propose and begin to test ways to confer resistance to the susceptible mosquitoes. Using the transposable element called Hermes, a tissue specific promoter from Drosophila, and the Green Fluorescent Protein as a genetic marker we engineered a strain of mosquitoes that expressed green fluorescence only in the flight muscles. Once a gene that confers resistance to the nematodes in mosquitoes, or is toxic only to the nematodes, is found the implications from this successful insect transformation are that this technology could be useful in combating human lymphatic filariasis.

Technical Abstract: A strategy to engineer a strain of Culex mosquitoes refractory to filarial transmission is described. A requirement for success of the strategy is identification of a flight muscle-specific promoter that functions in the mosquito. A green fluorescent protein (GFP) marker gene under the control of the promoter region of the Drosophila melanogaster act88F gene was inserted into the genome of Culex quinquefasciatus by Hermes-mediated germ-line transformation. Transformation was confirmed by classical Mendelian genetics: first generation transgenic mosquitoes were mated to wild-types, and second generation transgenics were sib-mated, resulting in progeny which expressed the dominant GFP gene in predicted Mendelian ratios for a single-locus integration event. Hybridization of a genomic Southern blot to a radiolabeled probe verified that the entire donor plasmid integrated into the mosquito genome. GFP expression in the transgenic mosquitoes was restricted to the adult flight muscles. Expression was distinctly more intense in homozygous animals, and the transgene was shown to be inserted near the sex locus. Implications regarding establishment of a homozygous strain, cloning of sex-related genes, and use of the D. melanogaster act88F promoter region to produce a genetically modified strain of Cx. quinquefasciatus refractory to development of filarial nematodes are discussed.

Last Modified: 7/28/2014
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