Submitted to: Meeting Abstract
Publication Type: Abstract Only
Publication Acceptance Date: 9/30/2007
Publication Date: N/A
Citation: N/A Interpretive Summary: N/A
Technical Abstract: Endoparasitic wasps often utilize symbiotic polydnaviruses (PDVs) as a means to overcome and regulate host development and immunity. Genetic manipulations and functional investigations of PDV immunosuppressive activities have been difficult due to the inability to replicate PDV DNA outside host wasps, the inability to maintain stable hemocyte and fat body cell cultures, and the inability to knockout or manipulate PDV gene expression in vivo. We have begun systematic evaluations of the Junonia coenia densovirus-based vectors pJDsRedMCSH, pJDsRedMCS-ES, and pJGFPMCSH (H. Bossin; P. Shirk, USDA, FL) as a means to overcome these limitations. The vectors express fluorescent markers (DsRed and GFP) and efficiently integrate into DNA of insect cells and embryos. We have developed a strategy to use the densovirus-derived vectors for stable delivery of PDV transgene cassettes to insect cell lines and Heliothis virescens embryos using the Campoletis sonorensis Ichnovirus (CsIV) vankyrin gene family and a yellow fluorescent protein (YFP) marker as models. RT-PCR analyses of Sf9 cells transfected with vectors incorporating CsIV vankyrins and YFP indicate that transgenes are expressed concurrently with vector-based DsRed and GFP markers within 24 hours. We have also generated and maintained polyclonal populations of Sf9 cells expressing the Dsred and GFP markers as well as YFP and vankyrin transgene cassettes. H. virescens embryos injected with the recombinant densovirus-based plasmids exhibit moderate hatch efficiencies (30-60%), but high transformation efficiencies with 70-80% of emerging larvae exhibiting DsRed marker expression primarily in the aorta and gut. Evaluations of transgene expression within transformed larvae are currently underway.