1a.Objectives (from AD-416):
Develop a screwworm strain designed such that only males are fed, reared, and processed, resulting in considerable savings realized by:(1) reduced levels of radiation for sterilization of males (mixed population of males and females requires higher doses of radiation to effect sterilization); (2) increased competitiveness of male-only strain, as compared to wild-type males, leading to a more effective eradication program (lower radiation dose reduces fitness losses in irradiated flies); (3) improved field identification of colony-produced/released screwworms genetically 'labeled' by the transgenic, male-only strain of screwworms' expression of the fluorescent protein; and (4) in the longer term, eliminating the need for sterilization prior to release by utilizing similar approaches to develop a genetically sterile male-only strain.
1b.Approach (from AD-416):
1. Further develop single component tetracycline-repressible female lethal conditional lethal vectors. Modify vectors to enhance female lethality by:
a) using blowfly gene promoters to boost tTAv expression; b) insertion of an additional promoter to co-express tTA and a cell death gene; c) insertion of a internal ribosome entry site for co-translation of tTAv and a cell death protein; and d) optimize codons in the tTAV to reflect C. hominivorax codon usage. This could promote higher levels of expression of tTAv and thus higher lethality.
2. Develop two component tetracycline-repressible female embryo lethal conditional lethal vectors. Isolate and characterize promoters from blowfly genes that are active early in development to ensure lethality occurs at the embryo stage. Use an activity assay to determine which promoter is the most active in embryos. Make several lines of each vector to identify which combinations give the highest level of female embryo lethality.
3. Rear transgenic strains in laboratory cages for several generations to evaluate the quality of resultant genetic sexing line(s) as they are developed. Determine the presence and chromosomal location of the transgene in randomly selected flies by PCR analysis of genomic DNA. Base one primer on the sequence of the flanking DNA and base the other primer on the left or right piggyBac end.
Monies from this agreement were used to develop the Assistance Type Cooperative Agreement between ARS and the NORTH CAROLINA STATE UNIVERSITY. Therefore, refer to the report for Project No: 6205-32000-035-02A; Accession No: 0423566 which is above.