Honors Theses
Date of Award
2009
Document Type
Undergraduate Thesis
Department
Chemistry and Biochemistry
First Advisor
Randy Wadkins
Relational Format
Dissertation/Thesis
Abstract
Topoisomerase I (TOPI) is a nuclear enzyme that relieves torsional strain in DNA during transcription or replication. Furthermore, TOPl is the target enzyme for camptothecin antitumor drugs. Traditional immunohistochemical methods of studying TOPl expression require cell fixation, which arrests cellular metabolism at an instance in time. In efforts where TOPl studies rely on a function of time, cell fixation must occur at many finite intervals so that researchers may take many data points to simulate a real time assessment. To study TOPl activity continuously in cells, we are implementing recent gene targeting/knock-in technology to create a natively expressed TOP 1-EGFP chimeric protein regulated by the TOPl endogenous promoter in an intact medulloblastoma cell line. Portions of the TOPl gene have been cloned in bacterial vectors. Vector sequences have been aligned with sequences from Genbank® assuring integrity. Currently, knock-in gene constructions are being made as templates for future knock-in and knockout events in the cell line to allow quick insertion of the modifications of interest. ATOPl-EGFP cell line will provide stable expression of a bioluminescentTOPl protein for collecting consistent, reproducible protein expression data for evaluating the targeting and efficacy of new camptothecin antitumor drugs.
Recommended Citation
Delancey, James Wayne, "Creation of a Topoisomerase I-EFGP Cell Line Using Recent Knock-in Technology" (2009). Honors Theses. 1978.
https://egrove.olemiss.edu/hon_thesis/1978
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