Title page for ETD etd-02072009-102134

Type of Document Master's Thesis
Author Traver, Brenna E
URN etd-02072009-102134
Title Exogenously-introduced Homing Endonucleases Catalyze Double-stranded DNA Breaks in Aedes aegypti
Degree Master of Science In the Life Sciences
Department Entomology
Advisory Committee
Advisor Name Title
Adelman, Zachary N. Committee Chair
Bloomquist, Jeffrey R. Committee Member
Myles, Kevin M. Committee Member
Tu, Zhijian Jake Committee Member
  • Homing endonuclease
  • Aedes aegypti
  • double-strand DNA break repair
Date of Defense 2009-01-13
Availability restricted
Aedes aegypti transmits the viruses which cause yellow fever, dengue fever, and dengue hemorrhagic fever. Homing endonucleases are selfish genetic elements which introduce double-stranded DNA (dsDNA) breaks in a sequence-specific manner. In this study, we aimed to validate a somatic assay to detect recombinant homing endonuclease (rHE)-induced dsDNA breaks in both cultured cells and adult female Ae. aegypti. While the cell culture-based two plasmid assay used to test rHE ability to induce dsDNA breaks was inconclusive, assays used to test rHEs in Ae. aegypti were successful. Recognition sequences for various rHEs were introduced into Ae. aegypti through germline transformation, and imperfect repair at each of these exogenous sites was evaluated. In mosquitoes containing a single exogenous HE site, imperfect gap repair was detected in 40% and 21% of clones sequenced from mosquitoes exposed to I-PpoI and I–SceI, respectively. In mosquitoes containing two exogenous HE sites flanking a marker gene (EGFP), 100% of clones sequenced from mosquitoes exposed to I-PpoI, I-CreI, and I-AniI demonstrated excision of EGFP. No evidence of EGFP excision or imperfect repair at any HE recognition site was detected in mosquitoes not exposed to a rHE. In summary, a somatic genomic footprint assay was developed and validated to detect rHE or other meganuclease-induced site-specific dsDNA breaks in chromosomal DNA in Ae. aegypti.
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