Submitted to: Meeting Abstract
Publication Type: Proceedings
Publication Acceptance Date: 9/19/2003
Publication Date: 11/15/2003
Citation: SHIRK, P.D., FURLONG, R.B., BOSSIN, H. DELETION OF DENSOVIRAL SEQUENCES REDUCES THE EFFICIENCY OF SOMATIC INTEGRATION OF JCDNV-DERIVED PLASMIDS IN INSECTS. MEETING ABSTRACT. 2003. www.insectscience.org/3.36
Technical Abstract: Plasmids containing the genome of the Junonia coenia lepidopteran densovirus (JcDNV) can be used to achieve somatic transformation that is stably maintained by integration into the genome of whole insects following microinjection into syncytial embryos (Royer et al, 2001) or by transfection into cells lines (Bossin et al, Submitted). We assessed the effect of sequence modifications including different expression cassettes on the efficiency of JcDNV plasmid somatic transformation activities in Lepidoptera and Diptera. The plasmid pJDsRed[3xP3EGFP]H, which contains the Densovirus P9 promoter controlling expression of DsRed fluorescent protein and the 3xP3EGFP expression cassette (Berghammer et al., 1999), was injected into syncytial embryos of the fruitfly Drosophila melanogaster, and the moths Plodia interpunctella, Ephestia kuehniella and Trichoplusia ni and the beetle, Tribolium castaneum. Somatic transformation using pJDsRed[3xP3EGFP]H was observed on the basis of either DsRed and/or GFP fluorescence in G0 embryos and larvae of all species at rates from 40-95%. In Drosophila, the expression pattern of GFP was consistent with germline transformed insects. Cloning of 3xP3EGFP in the unique PvuI site outside the JcDNV sequence did not affect the rate of somatic transformation. Removal of the JcDNV coding sequences for some of the nonstructural proteins or the right inverted terminal repeat had no effect on the rate of somatic transformation. Serial removal of JcDNV fragments showed that the somatic transformation activity depended upon a 470 bp region within the 3' terminas of the nonstructural protein coding sequence. These modifications demonstrate that the somatic transformation activity is dependent upon left inverted terminal repeat as well as sequences internal to the densovirus sequence in insects.