Project Number: 6615-22000-021-08
Project Type: Reimbursable

Start Date: Sep 01, 2003
End Date: Aug 31, 2007

Objective:
The specific objectives for this project are i) to assess parameters of fitness and stability of transgenic Mediterranean fruit fly genetic sexing strains expressing EGFP or DsRed under different conditions of rearing stress, including routine mass rearing protocols, and to assess their overall mating competitiveness under contained green-house conditions using established quality control protocols; ii) to assess the potential for intra-genomic mobilization of the piggyBac transgene in host strains by embryonic excision assays, and to assess transgene stability in the presence of mobile and non-mobile sources of transposase; iii) to assess inter-genomic transmission of autonomous and non-autonomous piggyBac vectors from medfly host strains to symbiont bacterial populations; and iv) to test new vectors with enhanced stability properties conferred by post-integration recombination resulting in deletion or inversion of transposon sequences required for mobility.

Approach:
The experimental approach to this project first involves the creation and molecular analysis of the transgenic medfly genetic sexing strains that will be the subject of risk assessment for their eventual use in release programs. To appreciate the significance of experimental approaches designed to assess the influence of mobilizing systems, a thorough analysis must be made of transgenic strain stability and fitness, as well as transgene stability under several rearing regimes. Existing transgenic strains and new strains created by piggyBac germ-line transformation will be analyzed on a molecular level to determine the number of vector integrations by Southern blot hybridization, their insertion site sequence by inverse PCR, and their genomic location by chromosomal in situ hybridization The existence of endogenous genomic systems that might act to mobilize a non-autonomous piggyBac vector will be determined by embryonic plasmid-based excision assays. Small-scale laboratory regimes will then be used to test the influence of introduced sources of instability on transgene stability within a population by introducing medflies that carry mobile and non-mobile sources of piggyBac transposase. The most serious risk for transgenic insect release is the potential for inter-genomic transgene movement into closely associated organisms. This will be initially tested by assessing mobile and non-mobile transgene movement from the medfly into symbiont and other bacteria normally present in host strains throughout their development. Finally, experiments will be initiated to test new piggyBac vectors that can be immobilized subsequent to their transposon-mediated integration into the genome.