Project Number: 6615-22000-021-14
Project Type: Reimbursable

Start Date: Jan 01, 2007
End Date: Jan 14, 2011

Objective:
1) To test the stabilization vector system in the Mediterranean fruit fly, Ceratitis capitata, by modifying the pBac{L1-PUbDsRed-L2-3xP3ECFP-R1} vector originally used in Drosophila. 2) To create new target site and donor vectors for efficient use in tephritid flies. This will include i) modification of the pBac{3xP3-FRT-ECFP-linotte-FRT3} target acceptor vector and the pSL-FRT-EYFP-linotte-FRT3 donor vector by replacing 3xP3 promoters with polyubiquitin promoters and removal of the linotte sequence, and ii) introducing new heterospecific FRT and loxP recombinations sites for repetitive insertions into target sites. 3) To test the modified RMCE target vector and stabilization system in C. capitata in vivo and in cell culture. 4) To create a series of target site strains in C. capitata and the Mexican fruit fly, Anastrepha ludens, that will be characterized initially in terms of strain fitness and transgene marker expression. Optimal strains will be genetically mapped with the target site molecularly characterized.

Approach:
Vectors for transformant stabilization and targeting developed for Drosophila will be modified by promoter sequence replacement and insertion of new recombination sites, with subsequent tested by germ-line transformation in tephritid host strains and transfection into tephritid cell lines. Host strains will be analyzed by insertion site sequencing, marker expression, and strain viability and fitness. Targeting by recombinase-mediated cassette exchange will be testing by providing donor vectors and recombinase helper vectors and testing for recombination events.