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Date of Award
4-21-2000
Document Type
Restricted Thesis: Campus only access
Degree Name
Bachelor of Science
Department
Biology
First Advisor
Valerie M. Kish
Abstract
The transcription factor P53 acts as a tumor suppressor protein and is known to be mutant in close to one-third of glioblastoma tumors. As this is lower than the 50% p53 mutation rate in other cancers, it appears that other factors may inactivate p53 even though it may be genotypically normal. To assess such functionality in regards to its genotype within a specific cell line, we have begun the development of a p53 functional assay. Cellular RNA was appropriately isolated from RT2 rat glioma cell lines and exclusively p53 DNA was amplified. This p53 DNA, after the addition of poly-adenosine nucleotides to its 3' ends, was ligated into the pCR3.1 vector. The final concentration of p53 DNA after addition of the 3'A's was 7.025ng/μl. The vector-insert complex was then incorporated into E. coli cells for replication. These cells were subsequently grown up and analyzed for the presence of p53 insert. The p53 insert could fit into the vector in one of two directions, and of 15 colonies randomly chosen, nine had insert in the sense direction and three in the anti-sense direction. Sequence analysis of five sense clones produced highly inconclusive results, thus new primers have been developed to secure complete sequencing of the entire RT2 p53 gene before performing the functional assay.
Recommended Citation
Springer, Jeffrey R., "Initial steps in the development of a p53 functional assay for use in RT2 glioblastoma cell line" (2000). Honors Theses. 523.
https://scholarship.richmond.edu/honors-theses/523