Exogenously-introduced Homing Endonucleases Catalyze Double-stranded DNA Breaks in Aedes aegypti

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dc.contributor.authorTraver, Brenna E.en
dc.contributor.committeechairAdelman, Zachary N.en
dc.contributor.committeememberBloomquist, Jeffrey R.en
dc.contributor.committeememberMyles, Kevin M.en
dc.contributor.committeememberTu, Zhijian Jakeen
dc.contributor.departmentEntomologyen
dc.date.accessioned2014-03-14T21:28:45Zen
dc.date.adate2009-02-26en
dc.date.available2014-03-14T21:28:45Zen
dc.date.issued2009-01-13en
dc.date.rdate2009-02-26en
dc.date.sdate2009-02-07en
dc.description.abstractAedes 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 IĆ¢ 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.en
dc.description.degreeMaster of Science in Life Sciencesen
dc.identifier.otheretd-02072009-102134en
dc.identifier.sourceurlhttp://scholar.lib.vt.edu/theses/available/etd-02072009-102134/en
dc.identifier.urihttp://hdl.handle.net/10919/40967en
dc.publisherVirginia Techen
dc.relation.haspartBETraver.pdfen
dc.rightsIn Copyrighten
dc.rights.urihttp://rightsstatements.org/vocab/InC/1.0/en
dc.subjectHoming endonucleaseen
dc.subjectAedes aegyptien
dc.subjectdouble-strand DNA break repairen
dc.titleExogenously-introduced Homing Endonucleases Catalyze Double-stranded DNA Breaks in Aedes aegyptien
dc.typeThesisen
thesis.degree.disciplineEntomologyen
thesis.degree.grantorVirginia Polytechnic Institute and State Universityen
thesis.degree.levelmastersen
thesis.degree.nameMaster of Science in Life Sciencesen

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