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Date of Award
Bachelor of Science
The mosquito Aedes aegypti is a vector for several human pathogens that cause infectious diseases such as dengue fever, chikungunya, yellow fever, and zika. This animal has evolved a preference to blood feed from humans, making it a cause of high medical concern. A method often used to study the molecular and neural basis of behavior in model organisms such as Drosophila melanogaster is the GAL4/UAS system, which employs the yeast transcription factor gene GAL4 and its associated UAS binding sites to drive expression of effector transgenes. This technique is known for its versatility, but there are limits to what it can reveal if position effects on expression are not properly controlled. When a transgene is randomly inserted into the genome, nearby suppressors or enhancers of endogenous gene expression influence the transgene. Therefore, each site in the genome has a different effect on a given transgene’s expression. To control for this “position effect”, we need to insert transgenes into the same location. The main objective of this work is to insert an attP recombination site into a number of A. aegypti eye pigment genes, to create docking strains for transgene integration that control for position effects and have visually recognizable phenotypes. An oligo containing homologous sequences and the attP site are co-injected into embryos with CRISPR/Cas9, which makes a double stranded break to disrupt the eye pigment genes white (w), scarlet (St), pink (P), cinnabar (Cn), sepia (Se) and ruby (Rb) and produce their respective eye phenotype. Once the attP is in place, any effector transgene can be inserted into the genome through recombination with a plasmid- bound attB site, causing the subsequent descendants to exhibit the same level of expression of that construct. A mixture containing the designed sgRNA, Cas9 protein, the attP oligo and water, was injected into pre-blastoderm embryos. DNA extraction and fragment analysis were used to identify the attP insertion in the mosaic G0’s. The attP site was also detected in later generations for the genes White and Scarlet. Once characterized, the docking lines will be used to introduce a UAS-luciferase construct into the mosquito genome. The level of chemiluminescence will serve as an indication of the expression of the UAS and whether position effects are controlled. This work represents a new approach to understand mosquito host detection at the molecular and cellular level.
Corsi, Ileana Daniela, "Optimizing the Genetic Toolkit for Aedes Aegypti Using Targeted Integration of Recombination Sites" (2016). Department of Biological Sciences - Undergraduate Honors Theses. 69.
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