Creating Isogenic Cell Cultures for Functional Studies of DNA Mismatch-Repair Public Deposited

http://ir.library.oregonstate.edu/concern/undergraduate_thesis_or_projects/k930bz96v

Descriptions

Attribute NameValues
Creator
Abstract or Summary
  • Colorectal cancer (CRC), the third leading cause of cancer death in the USA, depends on early detection for patient survival. Early detection is improved with the identification of high risk individuals. Risk of CRC development is a complex interaction between an individual’s genetics and environmental exposures. Polycyclic Aromatic Hydrocarbons (PAHs) are ubiquitous, priority pollutants formed from the incomplete combustion of carbon. 96% of PAH exposure is through the diet for non-smokers. Metabolism of PAHs can form diol epoxides capable of binding to DNA, causing bulky DNA adducts which can lead to mutations and cancer. Preliminary data suggest that DNA mismatch repair (MMR) modulates mutagenicity of PAHs. Successful MMR depends on the formation of the MutSα protein complex, a heterodimer of MSH2 and MSH6 proteins. The objective of this project was to create a model system with which to perform functional assays for MMR capacity. I hypothesized that transfecting MMR-proficient cells with siRNA (short interfering RNA) would be a useful approach for creating isogenic cell cultures with MMR deficiencies without preexisting mutations or high spontaneous mutation rates. A MMR-proficient cell line, HeLa, was transfected with siRNA specific for the MLH1 and MSH2 genes. Analysis of anti-MLH1 and anti-MSH2 transfected cultures through semi-quantitative immunoblotting demonstrated a reduction in protein accumulation of MLH1, MSH2 and MSH6 respectively between 48 and 72 hours post-transfection, relative to the untransfected and negative-control-transfected cells. To determine the effect of reduced MSH2 protein accumulation on the MMR capacity of cells post-transfection I measured the survival of cells after exposure to the purine analog 6-Thioguanine (6-TG). Cells transfected with anti-MSH2 siRNA did not respond as expected and demonstrated no significant difference in survival between the anti-MSH2 transfected cultures and cultures transfected with Allstars Negative control siRNA or the untransfected cultures.
License
Resource Type
Date Available
Date Issued
Degree Level
Degree Name
Degree Field
Degree Grantor
Commencement Year
Advisor
Non-Academic Affiliation
Subject
Rights Statement
Funding Statement (additional comments about funding)
Publisher
Peer Reviewed
Language
Replaces
Additional Information
  • description.provenance : Made available in DSpace on 2016-01-06T21:28:11Z (GMT). No. of bitstreams: 2Sarver_Thesis_2015_PDF.pdf: 1218725 bytes, checksum: 0436a2bb0162cc769cb6eefac525d7aa (MD5)Sarver_final seminar_2015.pptx: 4771380 bytes, checksum: 1bb835cf368866692fcbbb9a4795b96d (MD5)
  • description.provenance : Submitted by Wanda Crannell (brr@oregonstate.edu) on 2016-01-01T00:06:47ZNo. of bitstreams: 2Sarver_Thesis_2015_PDF.pdf: 1218725 bytes, checksum: 0436a2bb0162cc769cb6eefac525d7aa (MD5)Sarver_final seminar_2015.pptx: 4771380 bytes, checksum: 1bb835cf368866692fcbbb9a4795b96d (MD5)
  • description.provenance : Approved for entry into archive by Patricia Black(patricia.black@oregonstate.edu) on 2016-01-06T21:28:11Z (GMT) No. of bitstreams: 2Sarver_Thesis_2015_PDF.pdf: 1218725 bytes, checksum: 0436a2bb0162cc769cb6eefac525d7aa (MD5)Sarver_final seminar_2015.pptx: 4771380 bytes, checksum: 1bb835cf368866692fcbbb9a4795b96d (MD5)

Relationships

Parents:

This work has no parents.

Last modified

Downloadable Content

Download PDF

Items