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dc.contributor.authorAhmad, Alyaen_US
dc.date.accessioned2016-05-12T14:57:34Z
dc.date.available2016-05-12T14:57:34Z
dc.date.issued2015
dc.identifier.urihttps://hdl.handle.net/2144/16247
dc.description.abstractDNA repair is not a single mechanism found within cells. There exists numerous different DNA repair mechanisms that function within every type of cell. The majority of these mechanisms risk accumulating mutations. However, there are a few repair mechanisms that are known to be error-free and one of these is direct reversal repair. This study focused on two proteins highly involved in direct reversal DNA repair--ALKBH2 and ALKBH3. Previous studies have shown that in mice, these two proteins play a significant role in preventing and repairing DNA damage due to methylation as well as decreasing the frequency of mutagenic alkyl adducts. The goal of this study was to characterize the roles of the direct reversal repair proteins in human cells. We expected to see a similar phenotype to that of the Alkbh2 and Alkbh3-deficient mice. Telomerase immortalized human skin fibroblasts were targeted for the ALKBH2 and ALKBH3 alleles using a RNA-guided CRISPR-Cas9 construct that was designed to induce double stranded DNA breaks within the exons and disrupt the open reading frame, eliminating protein activity. Isolated clones were analyzed using fragment analysis and DNA sequencing to characterize any alterations in the open reading frame of the genes. Through sequencing analysis, results showed that one clone was successfully targeted for one of the ALKBH3 alleles with a single nucleotide insertion in its sequence, causing a disruption of the open reading frame. Though the ultimate goal of the experiment was not attained, we concluded that HTERTG fibroblasts can be expanded to serve as a model in which to construct targeted human cell lines that have near normal karyotypes.en_US
dc.language.isoen_US
dc.subjectCellular biologyen_US
dc.subjectALKBHen_US
dc.subjectAlkylation damageen_US
dc.subjectDirect reversal repairen_US
dc.subjectDNA repairen_US
dc.titleHuman targeted deletions and biological roles of genes involved in repair of alkylation damageen_US
dc.typeThesis/Dissertationen_US
dc.date.updated2016-04-08T20:19:40Z
etd.degree.nameMaster of Scienceen_US
etd.degree.levelmastersen_US
etd.degree.disciplineMedical Sciencesen_US
etd.degree.grantorBoston Universityen_US


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