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United States Department of Agriculture

Agricultural Research Service

Research Project: BIOLOGICALLY-BASED TECHNOLOGIES FOR MANAGEMENT OF CROP INSECT PESTS IN LOCAL AND AREA-WIDE PROGRAMS Title: Single inverted terminal repeats of the Junonia coenia Densovirus promotes somatic chromosomal integration of vector plasmids in insect cells and supports high efficiency expression

Authors
item Shirk, Paul
item Furlong, Richard

Submitted to: Meeting Abstract
Publication Type: Abstract Only
Publication Acceptance Date: June 12, 2009
Publication Date: June 12, 2009
Citation: N/A

Technical Abstract: Plasmids that contain a disrupted genome of the Junonia coenia densovirus (JcDNV) integrate into the chromosomes of the somatic cells of insects. When subcloned individually, both the P9 inverted terminal repeat (P9-ITR) and the P93-ITR promote the chromosomal integration of vector plasmids in insect embryonic cells. Microinjection of plasmids that included the 517 bp of either the P9-ITR or the P93-ITR into embryos of Drosophila melanogaster resulted in somatically transformed larvae and adults with efficiencies comparable to the JcDNV vectors. A somatic transformation vector, pDP9, that includes the P9-ITR was constructed. The pDP9 vectors provide a simplified means of producing somatically transformed larvae or permanently transformed insect cells. Transformation of Bombyx mori Bm5 cells or Spodoptera frugiperda Sf9 cells with pDP9 vectors result in the integration of the pDP9 vectors into genomic DNA of the Bm5 and Sf9 cells. In addition, the pDP9 vectors support high levels of expression of foreign genes. Insertion of foreign genes 3’ of the densoviral P9 promoter results in a high level production of the gene by pDP9 transformed cells. P9 driven transcription within the pDP9 transformed Sf9 cells results in foreign gene transcript levels 5,000 fold higher than constitutively expressed actin 3 genes. The pDP9 vector transformation results in the efficient selection of high-producing clones for biosynthesis of target proteins.

Last Modified: 12/22/2014
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