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ARS Home » Southeast Area » Gainesville, Florida » Center for Medical, Agricultural and Veterinary Entomology » Insect Behavior and Biocontrol Research » Research » Publications at this Location » Publication #321796

Research Project: Biologically-based Technologies for Management of Crop Insect Pests in Local and Areawide Programs

Location: Insect Behavior and Biocontrol Research

Title: Insect cell transformation vectors that support high level expression and promoter assessment in insect cell culture

Author
item Shirk, Paul
item Furlong, Richard

Submitted to: Plasmid Journal
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 1/11/2016
Publication Date: 1/22/2016
Citation: Shirk, P.D., Furlong, R.B. 2016. Insect cell transformation vectors that support high level expression and promoter assessment in insect cell culture. Plasmid Journal. 83:12-19. Available: http://dx.doi.org/10.1016/j.plasmid.2016.01.001

Interpretive Summary: The biotechnology necessary to assess gene regulation is a critical component of developing strategies for alternative methods of control for pest insects. Scientists at the USDA, Agriculture Research Service, Center from Medical, Agricultural and Veterinary Entomology, Gainesville, Florida, have identified a region in the genome of an insect virus that controls the integration of the viral DNA sequences into host cell chromosomes. The virus DNA sequence was used to construct new gene vector system that can be used for cell transformation and high level expression in cultured insect cells. The use of these vectors provides a valuable source to test gene promoter systems for the development of alternative methods of pest control.

Technical Abstract: A somatic transformation vector, pDP9, was constructed that provides a simplified means of producing permanently transformed cultured insect cells that support high levels of protein expression of foreign genes. The pDP9 plasmid vector incorporates DNA sequences from the Junonia coenia densovirus that are involved in integration of the densovirus in insect cell chromosomes and a promoter/enhancer system that results in high levels of expression. The plasmid also contains two markers that permit selection of transformed insect cells by antibiotic resistance or by cell-sorting for fluorescent protein expression. Transformation of Bombyx mori Bm5 or Spodoptera frugiperda Sf9 cultured cells with the pDP9 vectors results in the integration of the pDP9 plasmid into genomic DNA of Bm5. pDP9 contains a multiple cloning site (MCS) 3’ of the densoviral P9 promoter and insertion of a protein coding sequence within the MCS results in high level expression by pDP9 transformed cells. P9 driven transcription in the pDP9 transformed Sf9 cells produced foreign gene transcript levels that were 30 fold higher than actin 3 driven transgenes and equivalent to hr5IE1 driven transgenes. The pDP9 vector transformation results in the efficient selection of clones for assessment of promoter activity.