Mosquito-borne infectious diseases continue to have significant impact on the health and economy of much of the world. Alternative strategies for control of mosquito vectors could arise through a greater understanding of the genetics and physiology of these insects. Progress in genetic research with mosquitoes will be greatly enhanced by the development of effective and reproducible means of transgenic manipulations. A key component of this capability is the identification and manipulation of insect transposons that are able to efficiently vector genes into the mosquito genome, permit gene tagging, and maintain genes in an active state. The convergence of research in this field has resulted in the successful transgenic manipulation of mosquitoes with several insect transposons, each with its own unique properties. The insect transposon piggyBac has been demonstrated to be effective for genetic manipulation in widely diverse insect species, including mosquitoes. Recently, we demonstrated transformation of Aedes acgypti with piggyBac using the cinnabar eye coloration marker gene. This proposal seeks significant funding to understand and exploit the capabilities of the piggyBac transposon for establishing and maintaining transgenic strains of Aedine mosquito species. Additional piggyBac-based transgene vectors containing desirable genes under the control of either ubiquitous insect promoters or tissue-specific mosquito promoters will be constructed, tested, and combined with the selectable markers for transgenesis. The presence and expression of the transposon and vectored genes will be detected following selection for the co-transforming marker gene at the Gi generation, and analyzed using a variety of techniques including Southern hybridization, PCR amplification and sequencing, Northern and Western analyses, and immunoflourescence, as appropriate. Genetic characterization of the stability and movement of transgene sequences within the mosquito genome will be examined over several generations. This research will provide an exciting and much needed capability for significant enhancement of the basic research effort in mosquito genetics and physiology.

National Institute of Health (NIH)
National Institute of Allergy and Infectious Diseases (NIAID)
Research Project (R01)
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Special Emphasis Panel (ZRG1-TMP (01))
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Aultman, Kathryn S
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University of Notre Dame
Schools of Arts and Sciences
Notre Dame
United States
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Nawtaisong, Pruksa; Fraser, Mark E; Carter, James R et al. (2015) Trans-splicing group I intron targeting hepatitis C virus IRES mediates cell death upon viral infection in Huh7.5 cells. Virology 481:223-34
Franz, Alexander W E; Balaraman, Velmurugan; Fraser Jr, Malcolm J (2015) Disruption of dengue virus transmission by mosquitoes. Curr Opin Insect Sci 8:88-96
Carter, James R; Keith, James H; Fraser, Tresa S et al. (2014) Effective suppression of dengue virus using a novel group-I intron that induces apoptotic cell death upon infection through conditional expression of the Bax C-terminal domain. Virol J 11:111
Carter, James R; Balaraman, Velmurugan; Kucharski, Cheryl A et al. (2013) A novel dengue virus detection method that couples DNAzyme and gold nanoparticle approaches. Virol J 10:201
Carter, James R; Keith, James H; Barde, Pradip V et al. (2010) Targeting of highly conserved Dengue virus sequences with anti-Dengue virus trans-splicing group I introns. BMC Mol Biol 11:84
Chompoosri, Jakkrawarn; Fraser, Tresa; Rongsriyam, Yupha et al. (2009) Intramolecular integration assay validates integrase phi C31 and R4 potential in a variety of insect cells. Southeast Asian J Trop Med Public Health 40:1235-53
Nawtaisong, Pruksa; Keith, James; Fraser, Tresa et al. (2009) Effective suppression of Dengue fever virus in mosquito cell cultures using retroviral transduction of hammerhead ribozymes targeting the viral genome. Virol J 6:73
Keith, James H; Schaeper, Cheryl A; Fraser, Tresa S et al. (2008) Mutational analysis of highly conserved aspartate residues essential to the catalytic core of the piggyBac transposase. BMC Mol Biol 9:73
Keith, James H; Fraser, Tresa S; Fraser Jr, Malcolm J (2008) Analysis of the piggyBac transposase reveals a functional nuclear targeting signal in the 94 c-terminal residues. BMC Mol Biol 9:72
Lobo, Neil F; Fraser, Tresa S; Adams, John A et al. (2006) Interplasmid transposition demonstrates piggyBac mobility in vertebrate species. Genetica 128:347-57

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