Development of Novel Plasmids for use in Neuroregeneration Studies in Zebrafish
Dr. Leah Chase
Nicole Ladd, Dr. Brent Krueger, Hope College Department of Chemistry and Dr. Aaron Putzke, Whitworth University Department of Biology
System xc– is an amino acid transporter that enables the transfer of intracellular glutamate in exchange for extracellular cystine. This process is involved in reducing oxidative stress within cells. This transporter is commonly found in the central nervous system, most commonly in neuroprotective cells such as astrocytes and glia. Mitigation of oxidative stress is thought to be critical to neurogeneration of the central nervous system. Neuroregeneration is a trait found in zebrafish and other teleost fishes, and can be observed following neuronal ablation via laser. In order to examine the role system xc– plays in this process, our project aims to generate novel plasmids for use in zebrafish lines, allowing for the fluorescent expression of xc– in the nervous system. The three plasmids we are creating are hb9:hsp:mCHERRY, hb9:hsp:XCT-EGFP, and GFAP:XCT-EGFP. These can be inserted into zebrafish and the lines grown, to enable studies aimed at understanding System xc– and the neuroregeneration process. The hb9:hsp:mCHERRY plasmid will allow motor neurons to specifically be stained red in our zebrafish so that they can be targeted for neuronal ablation. The hb9:hsp:XCT-EGFP will allow for the expression of a green fluorescent-tagged System xc- transporter only in motor neurons so that we can directly observe how System xc– expression and trafficking changes during the neuroregeneration process. Finally, the GFAP:XCT-EGFP will allow for selective expression of a green-fluorescent form of System xc– in glial cells to determine if these cells also contribute to the regeneration process.
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