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dc.contributor.advisorKnutsen, Erik
dc.contributor.authorRosenlund, Ingrid Arctander
dc.date.accessioned2021-06-03T05:54:12Z
dc.date.available2021-06-03T05:54:12Z
dc.date.issued2019-06-03en
dc.description.abstractThe aim of this project was to create a stable knockout cell model with the CRISPR/Cas9 method for programmable genome editing that would make it possible to run long term assays to further investigate the cellular function of the long non-coding RNA FAM83H-AS1. The CRISPR/Cas9 method is compared to gene knockdown with small interfering RNAs (siRNAs) and short hairpin RNAs (shRNAs) via the RNA interference (RNAi) system in cells. The cell lines T-47D, MDA-MB-468, and BT474 were transfected with the CRISPR/Cas9 components by electroporation and single cell sorted by flow cytometry. Wild type T-47D cells were transfected with siRNAs towards the target gene. knockout status of the CRISPR clones was determined by PCR, and the expression levels of FAM83H-AS1 and FAM83H was measured with RT-qPCR. Our attempt at creating a CRISPR knockout cell line resulted in eight T-47D CRISPR clones; three heterozygous knockout clones, and five clones where the FAM83H-AS1 gene remained intact in both alleles. We were not successful at making a homozygous knockout clone, or expanding CRISPR clones in MDA-MB-468 and BT474 cells. Analyses of the T-47D clones with RT-qPCR showed variable expression levels of FAM83H-AS1 in the three heterozygous clones. The expression level of FAM83H seems to follow the expression of FAM83H-AS1 in all clones. This was not seen with siRNA knockdown of FAM83H-AS1, and suggests a relationship between the sense, FAM83H, and antisense gene, FAM83H-AS1, at the transcriptional level. Conclusively, there are several advantages and disadvantages with each knockdown strategy, and as our results show the CRISPR/Cas9 method is not the most suitable option for long term knockout of gene expression in our epithelial cell lines. Knockdown with shRNA might prove to be a feasible alternative as stable gene silencing is possible and some of the methodological issues with the CRISPR/Cas9 technique is overcome.en_US
dc.identifier.urihttps://hdl.handle.net/10037/21334
dc.language.isoengen_US
dc.publisherUiT Norges arktiske universitetno
dc.publisherUiT The Arctic University of Norwayen
dc.rights.holderCopyright 2019 The Author(s)
dc.rights.urihttps://creativecommons.org/licenses/by-nc-sa/4.0en_US
dc.rightsAttribution-NonCommercial-ShareAlike 4.0 International (CC BY-NC-SA 4.0)en_US
dc.subject.courseIDMED-3950
dc.subjectVDP::Medical disciplines: 700::Basic medical, dental and veterinary science disciplines: 710::Medical molecular biology: 711en_US
dc.subjectVDP::Medisinske Fag: 700::Basale medisinske, odontologiske og veterinærmedisinske fag: 710::Medisinsk molekylærbiologi: 711en_US
dc.titleExperimental gene expression modulation - A practical and theoretical approachen_US
dc.typeMaster thesisen
dc.typeMastergradsoppgaveno


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Attribution-NonCommercial-ShareAlike 4.0 International (CC BY-NC-SA 4.0)
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