Department

Department of Molecular Biology

First Advisor

Stephen L. Denton

Second Advisor

Jason P. Gigley

Description

Toxoplasma gondii is a eukaryotic parasite that can infect a wide array of hosts multiple times and cannot be eradicated. Effects can lead to blindness, brain inflammation, and fatality in immunocompromised patients by developing toxoplasmosis. By understanding the nature of Toxoplasma gondii infection, future research could lead to treatments or complete removal from the host. The parasite invades a host cell by specialized organelles secreting proteins called rhoptries, micronemes, and dense granules that facilitate active invasion. Mechanisms of parasitic lifestyle such as replication, immune invasion, and nutrient acquisition within the host cell are not fully characterized. To understand genes involved with these aspects of fitness, a reverse genetic screen was employed. A double stranded break was introduced at targeted sites of the genome using CRISPR-CAS9, which causes mutations as the parasite attempts to repair the induced double stranded break. To select for these mutants, we hijack the repair mechanisms to insert a resistance cassette in place of our targeted gene, resulting in a null mutation. We asses contribution of fitness of the selected gene by monitoring growth, replication, motility, and invasion. After analysis of genes involved with parasite fitness, these gene candidates will lead to future research into the mechanisms they are involved with.

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Reverse Genetic Screen in Toxoplasma gondii

Toxoplasma gondii is a eukaryotic parasite that can infect a wide array of hosts multiple times and cannot be eradicated. Effects can lead to blindness, brain inflammation, and fatality in immunocompromised patients by developing toxoplasmosis. By understanding the nature of Toxoplasma gondii infection, future research could lead to treatments or complete removal from the host. The parasite invades a host cell by specialized organelles secreting proteins called rhoptries, micronemes, and dense granules that facilitate active invasion. Mechanisms of parasitic lifestyle such as replication, immune invasion, and nutrient acquisition within the host cell are not fully characterized. To understand genes involved with these aspects of fitness, a reverse genetic screen was employed. A double stranded break was introduced at targeted sites of the genome using CRISPR-CAS9, which causes mutations as the parasite attempts to repair the induced double stranded break. To select for these mutants, we hijack the repair mechanisms to insert a resistance cassette in place of our targeted gene, resulting in a null mutation. We asses contribution of fitness of the selected gene by monitoring growth, replication, motility, and invasion. After analysis of genes involved with parasite fitness, these gene candidates will lead to future research into the mechanisms they are involved with.