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ARS Home » Southeast Area » Gainesville, Florida » Center for Medical, Agricultural and Veterinary Entomology » Insect Behavior and Biocontrol Research » Research » Publications at this Location » Publication #333074

Research Project: Improved Biologically-Based Methods for Insect Pest Management of Crop Insect Pests

Location: Insect Behavior and Biocontrol Research

Title: Cre/lox-recombinase-mediated cassette exchange for reversible site-specific genomic targeting of the disease vector, Aedes aegypti

item HACKER, IRINA - Justus-Liebig University
item HARRELL II, ROBERT - University Of Maryland
item EICHNERD, GERRIT - Justus-Liebig University
item PILITT, KRISTINA - University Of Maryland
item O BROCHTA, DAVID - University Of Maryland
item Handler, Alfred - Al
item SCHETELIG, MARC - Justus-Liebig University

Submitted to: Scientific Reports
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 1/30/2017
Publication Date: 3/7/2017
Citation: Hacker, I., Harrell Ii, R., Eichnerd, G., Pilitt, K., O Brochta, D., Handler, A.M., Schetelig, M.F. 2017. Cre/lox-recombinase-mediated cassette exchange for reversible site-specific genomic targeting of the disease vector, Aedes aegypti. Scientific Reports. 7:43883.

Interpretive Summary: The control of arthropod diseases of agricultural and medical importance, especially those vectored by mosquitoes, is a critical emphasis for USDA. In order to construct new biological control tools for mosquitoes, methods for creation of high-efficiency, site-targeted genetically modified insect strains that lead to more effective biological control programs were developed. Researchers at the USDA, Agriculture Research Service, Center for Medical, Agricultural and Veterinary Entomology, Gainesville, Florida, in collaboration with national and international scientists at University of Maryland and Justus-Liebig-University Giessen, Germany, developed methods for the first successful test of a site-specific genome targeting system for the yellow fever mosquito. This vector system leads to the insertion of new genes that are no longer mobile and highly stable within the mosquito genome. It also avoids the insertion of unneeded or unwanted vector sequences, is reversible, and permits subsequent retransformation with multiple additional gene cassettes targeted to the same site. This system should provide a significant improvement for the development of stable and contained transgenic mosquito strains intended for field release. Mosquitoes and other agriculturally important pest insects derived from this method will provide a resource of a highly stable genetically altered insect that can be used to control outbreaks of pest populations.

Technical Abstract: Site-specific genome modification is an important tool for mosquito functional genomics studies that help to uncover gene functions, identify gene regulatory elements, and perform comparative gene expression studies, all of which contribute to a better understanding of mosquito biology and are thus a key to finding new strategies to eliminate vector-borne diseases. Moreover, it allows the creation of advanced transgenic strains that can be used to establish new or improve existing vector control programs. Site specific modification circumvents the drawbacks of transposon mediated transgenesis, where the random transgene integration into the host genome often results in reduced fitness due to insertional mutagenesis. In addition, variable position effects influence transgene expression. We chose the Cre/lox site-specific targeting system that very efficiently is used for recombinase mediated cassette exchange (RMCE) in Drosophila, and applied it for the first time to a mosquito, Aedes aegypti, the vector of dengue, chikungunya and Zika viruses. In this context we created several RMCE landing site lines and evaluated their fitness cost due to transgene integration. Cassette exchange was achieved in a two-step mechanism via a donor plasmid integration intermediate. Compared to the existing phiC31/att-RMCE system for Ae. aegypti, Cre/lox-RMCE has two advantages: 1) in contrast to att sites, the lox sites remain unaltered during recombination. Therefore, the same site can be targeted repeatedly, which allows successive modifications and quick expansion or adaptation of existing systems; 2) Cre/lox-RMCE has the benefit of being reversible, thus allowing the excision of undesired sequences.