Please use this identifier to cite or link to this item: http://hdl.handle.net/10790/5172

INTEGRATION OF A “SELF-DOCKING SITE” GENETIC CONSTRUCT IN THE SOUTHERN HOUSE MOSQUITO (CULEX QUINQUEFASCIATUS) AS A STEP TOWARD GENETIC CONTROL STRATEGIES

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Title:INTEGRATION OF A “SELF-DOCKING SITE” GENETIC CONSTRUCT IN THE SOUTHERN HOUSE MOSQUITO (CULEX QUINQUEFASCIATUS) AS A STEP TOWARD GENETIC CONTROL STRATEGIES
Authors:Nishimoto, Jared Hajime Ke'ale
Contributors:Sutton, Jolene (advisor)
Tropical Conservation Biology & Environmental Science (department)
Keywords:Genetics
Conservation biology
Molecular biology
Avian Malaria
Conservation
show 4 moreGene Drive
Hawaii
Mosquito
Underdominance
show less
Date Issued:Jul 2019
Abstract:Background: Since its initial introduction to Hawaiʻi, Culex quinquefasciatus, continues to threaten native birds by vectoring Plasmodium relictum; the parasite responsible for avian malaria. Avian malaria will become a larger threat with global warming expanding the range in which both the vector and disease can develop. Conventional methods to mitigate mosquito threats are not feasible on a large, landscape scale. Genetic modification of mosquito populations may be more efficient and longer lasting than smaller scale or more traditional mosquito control methods.
Study focus: We propose to create an underdominance-based gene drive system in Cx. quinquefasciatus. This gene drive system could transform wild mosquito populations in Hawaiʻi to carry a Plasmodium refractory gene, which will offer a new tool that could potentially mitigate the decline of native Hawaiian birds. In this study, we aimed to develop the first step in creating an underdominance-based gene drive system, which was to integrate and express a phiC31(ΦC31) integrase construct that includes a “self-docking site”. We used a Restriction Enzyme Mediated Integration (REMI) strategy to integrate a ΦC31integrase construct with a DsRed2 phenotype marker (a red florescent protein) into wildtype Cx. quinquefasciatus. Integration was confirmed via PCR and sequencing, establishing that REMI, which has never been used to genetically modify mosquitoes before, is a viable strategy for germline genetic modification in mosquitoes. However, due to problems with accurate screening of the DsRed2 phenotype, we also microinjected wildtype Cx. quinquefasciatus with an edited version of the ΦC31plasmid, called the “pBattP-EGFP” construct, to attempt to improve screening efficiency and to better assess efficacy of the use of REMI. This edited construct contained an alternative phenotype marker, a gene for a green florescent protein (EFGP), under control of a promoter (3xP3) that was expected to drive expression in the eye regions, for easier diagnostic screening. However, there was no success using the pBattP-EGFP construct to transform Cx. quinquefasciatus. Future research should focus on increasing transformation and screening efficiency by exploring alternative construct components and alternative genome editing methods.
Broader impacts: The ΦC31 plasmid is an important component because it contains a self-docking site, which will allow site-specific integration of additional genes of interest. Subsequent microinjections could include possible effector genes, such as an avian malaria refractory gene. The self-docking site can be used to attach any other type of refractory gene, such as a human West Nile Fever refractory gene, for which Cx. quinquefasciatus is a primary vector. Successful integration and expression of the ΦC31construct in Cx. quinquefasciatus will open up many different possibilities to control wild mosquito populations through the use of the self-docking site, whether it is to control for a human or animal disease.
Pages/Duration:59 pages
URI:http://hdl.handle.net/10790/5172
Appears in Collections: Tropical Conservation Biology and Environmental Science


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