Inactivation of tumor supressor gene BRCA1 causes a life-long risk of breast carcinoma development. Genetic screenings of indicated individuals from high-risk families help to identify large number of sequence variants in known predisposing genes. Majority of discovered variants doesn't have clinical significance yet which causes a big problem for diagnostics. Some of these variants are found within regulatory non-coding regions of gene. A part of the clinical classification of variants is their functional characterization. The goal of this thesis was to create a model system for functional characterization of variants in non-coding regions and to verify its function. Model system was based on targeted gene manipulation by co-transfecting CRISPR-Cas9 construct and donor construct that contained a portion of BRCA1 gene sequence with analyzed modifications, into U2 OS cells. The cells have stably integrated DR-GFP system which allows the activity of homologous recombination (HR) to be determined. Monoallelic modifications were induced into U2 OS cells. These modifications were in a Kozak sequence region of BRCA1 gene. Expression level of BRCA1 mRNA was determined by qRT-PCR, which showed the same levels of mRNA in all cells with analyzed alterations. Next, expression level of BRCA1 protein was...
| Identifer | oai:union.ndltd.org:nusl.cz/oai:invenio.nusl.cz:405676 |
| Date | January 2019 |
| Creators | Bubáková, Eliška |
| Contributors | Ševčík, Jan, Vodička, Pavel |
| Source Sets | Czech ETDs |
| Language | Czech |
| Detected Language | English |
| Type | info:eu-repo/semantics/masterThesis |
| Rights | info:eu-repo/semantics/restrictedAccess |
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