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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: Functionality of Jcdnv-Derived Somatic Transformation Vectors in Insects and the Role of Viral Enhancer Sequences

Authors
item Shirk, Paul
item Gillett, Jennifer - UNIV OF FL, GAINESVILLE
item Bossin, Herve - FAO/IAEA, SEIBERSDORF, AU

Submitted to: Journal of Insect Science
Publication Type: Proceedings
Publication Acceptance Date: December 30, 2006
Publication Date: December 18, 2006
Citation: Shirk, P.D., Gillett, J., Bossin, H. 2006. Functionality of JCDNV-Derived Somatic transformation vectors in insects and the role of viral enhancer sequences. Journal of Insect Science. 6:46.

Technical Abstract: Stable somatic transformation of insects following microinjection of syncytial embryos (Royer et al, 2001, J Virol. 77: 11060) or by transfection of cells lines (Bossin et al, 2003, Insect Mol. Biol. 10: 275) can be achieved by integration of entire plasmids containing the Junonia coenia lepidopteran densovirus (JcDNV) genome. We assessed effects of sequence modifications including the presence of expression cassettes on the efficiency of JcDNV somatic transformation activities in Lepidoptera and Diptera. Cloning of 3xP3EGFP outside the JcDNV sequence did not affect the somatic transformation rate. Removal of coding sequences for some JcDNV nonstructural proteins or the 3’ inverted terminal repeat (ITR) had no effect on the transformation rate. Removal of 177 bp from the 5’ ITR did not decrease somatic transformation rates. However, removal of a 680 bp region within the 3’ terminus of the nonstructural protein coding sequence eliminated most transcriptional activity directed by the P9 promoter. Addition of the 680 bp DNV-enhancer to JcDNV vectors lacking this sequence restored transcriptional activity. Together with previously published results, these modifications demonstrate that the somatic transformation activity is dependent upon sequences of the 3’ ITR and influenced by sequences internal to the densovirus genome.

Last Modified: 10/25/2014
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