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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 AREAWIDE PROGRAMS

Location: Insect Behavior and Biocontrol Research Unit

Title: FLP recombinase-mediated site-specific recombination in silkworm, Bombyx mori

Authors
item Long, Ding-Pei -
item Zhao, Ai-Chun -
item Chen, Xue-Jiao -
item Zhang, Yang -
item Lu, Wei-Jian -
item Guo, Qing -
item Handler, Alfred

Submitted to: PLoS One
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: June 1, 2012
Publication Date: June 29, 2012
Citation: Long, D., Zhao, A., Chen, X., Zhang, Y., Lu, W., Guo, Q., Handler, A.M. 2012. FLP recombinase-mediated site-specific recombination in silkworm, Bombyx mori. PLoS One. 7(6):e40150. doi: 10.1371/journal.pone.00401.

Interpretive Summary: The creation of transgenic strains of economically important insects for the development of more effective biological control programs, and to improve beneficial species, is a major goal of our laboratory at the USDA, Agriculture Researach Service, Center for Medical, Agricultural and Veterinary Entomology, Gainesville, Florida. Development of this methodology and strategies to effectively and safely utilize transgenic insects will depend upon new means of moving genes to specific sites and so avoid mutations and the less favorable locations. This article describes the development of such a new gene transfer vector. Optimal target strains can then be used for the efficient integration of additional new genes. This vector targeting system was successfully tested for the first time in the silkmoth and has the potential to be used in other economically important insect species.

Technical Abstract: A comprehensive understanding of gene function and the production of site-specific genetically modified mutants are two major goals of genetic engineering in the post-genomic era. Although site-specific recombination systems have been powerful tools for genome manipulation of many organisms, they have not yet been established for use in the manipulation of the silkworm Bombyx mori genome. In this study, we achieved site-specific excision of a target gene at predefined chromosomal sites in the silkworm using a FLP/FRT site-specific recombination system. We first constructed two stable transgenic target silkworm strains that both contain a single copy of the transgene construct comprising a target gene expression cassette flanked by FRT sites. Using pre-blastoderm microinjection of a FLP recombinase helper expression vector, 32 G3 site-specific recombinant transgenic individuals were isolated from five of 143 broods. The average frequency of FLP recombinase-mediated site-specific excision in the two target strains genome was approximately 3.5%. This study shows that it is feasible to achieve site-specific recombination in silkworms using the FLP/FRT system. We conclude that the FLP/FRT system is a useful tool for genome manipulation in the silkworm. Furthermore, this is the first reported use of the FLP/FRT system for the genetic manipulation of a lepidopteran genome and thus provides a useful reference for the establishment of genome manipulation technologies in other lepidopteran species.

Last Modified: 9/1/2014
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