This work will contribute to an understanding of the reproductive physiology of the mosquito, an important vector of human and animal diseases including malaria, yellow fever, dengue, encephalitis and others worldwide. The advent of recombinant DNA technology has raised the hope that the mosquito could be genetically engineered, with the aim of preventing transmission of disease-causing organisms. It is, therefore, vital that we understand how genes are regulated in this insect. This work will focus on the control of gene expression at the molecular level in a mosquito. A long term goal of the research is to contribute to the development of technology that will allow the genetic manipulation of mosquitoes. Genes that play a role in mosquito reproduction provide an essential tool for developing the molecular biology and genetics of this important pest insect. The present proposal is focused on female-specific genes that are expressed during egg development in the response to the steroid hormone, 20-hydroxyecdysone. We plan to build upon earlier studies in which we have identified, from genomic libraries, 4 clones encoding vitellogenin genes - which are expressed only in the fat body in response to ecdysone - and another clone, containing an ecdysone-inducible gene expressed specifically in ovarian follicle cells. An important goal of this proposal is to characterize the 5'- regulatory sequences of these genes, with the ultimate goal of developing constructs that can be used to define nucleotide regions that confer ecdysone responsiveness and tissue-specificity of gene expression. Since functional analysis of gene expression in mosquitoes requires efficient transformation techniques, the development of which has been severely hampered by the lack of cloned mosquito genes, a second major goal of this proposal is to optimize a mosquito transfection system to allow the rapid analysis of hormone-inducible gene constructs in vitro. It is clear that genetic transformation of mosquitoes is a reasonable goal given the powerful tools made available by recombinant DNA technology, and the rapid advances being made in the transformation of a wide variety of plants and animals. Given the obvious medical importance of the mosquito, the choice of this insect as a particular target for genetic transformation is apparent.
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