KSEF R&D Excellence: Somatic Cell Expression of Densovirus Vectors for Gene Over-Expression and Suppression

Genes are usually studied by suppressing or enhancing their expression. While this approach is powerful for investigation of model systems such as Drosophila melanogaster where methods for mutagenesis and over-expression are well developed, it is more difficult for investigations of other insect systems. Nevertheless, it is these non-model systems that are associated with many of our most significant problems and general methods for study of these insect systems are urgently needed. My laboratory has been interested in general approaches to manipulating gene expression in the context of studying function of an unusual group of insect viruses, the polydnaviruses. Polydnaviruses have dramatic effects on their insect hosts in that they inhibit growth, prevent development and suppress insect immunological responses. However, these physiological effects are most readily observed at the organismal level and the system does not enable production of mutant viruses. To circumvent this problem, we have investigated an insect virus that efficiently integrates into genomic DNA but is expressed only in somatic cells. We suspect that this densovirus somatic transformation vector will enable both overexpression of a gene of interest and selective inactivation of the gene if RNA is expressed that will fold back on itself to form dsRNA and thereby trigger RNAi responses. In this proposal, we seek support to establish this system by 1) transforming cell lines to express dsRNA, 2) constructing densovirus vectors that enable facile manipulation of genes of interest (alternative promoters, useful cloning sites etc.) and 3) establishing effective techniques for transforming non-model insect species. This technology has been proposed in a federal grant that was not funded because the technology was not fully demonstrated. Establishing the technology will not only support resubmission of this grant, but will provide a viral vector system with multiple applications and readily useful for the study of non-model insects.