Start Date: Aug 15, 2010
End Date: Aug 15, 2012
The project has advanced to the stage where transformation of C. hominivorax with the female conditional-lethal vectors are necessary to evaluate their potential for implementation in the Screwworm Eradication Program's facility at Pacora, Panama. These transformation must be done in the biological containment facilities at Pacora. This agreement will be used to provide research and technical needs to carry out the female lethal vector evaluations and develop a second vector as a contingency in the event the current vectors do not meet the Program's needs. To meet these project needs, the first half of the project will be conducted at the ARS-Kerrville facility, while the second half of the project will be conducted at the ARS-Panama laboratory in Pacora, Panama. Year 1 Approach: (at Knipling-Bushland U.S. Livestock Insects Research Laboratory) 1. Develop tetracycline-repressible female lethal conditional lethal cloning vector based on piggybac transposable element system, C. hominivorax transformer sex-specific splicing intron, tTAV coding region, and tetO operon/promoter. a. Using an available C. hominivorax expressed gene database, optimize codons in the tTAV to reflect C. hominivorax codon usage. b. Isolate the transformer sex-specific intron from C. hominivorax. c. Create new candidate female-lethal vector using the optimized tTAV coding region and C. hominivorax transformer intron. 2. Evaluate the new vector in Drosophila melanogaster. Test for gene expression and female lethality. Year 2 Approach: (at ARS Screwworm Research Unit laboratory in Pacora, Panama) 1. Perform microinjections of all available female conditional lethal vector constructs into C. hominivorax and evaluate for female-specific lethality. 2. Provide technical advice and participate in transgenic strain rearing and maintenance.