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dc.contributor.advisorHaugen, Aage
dc.contributor.advisorMollerup, Steen
dc.contributor.advisorBersaas, Audun Trygge Haugen
dc.contributor.authorFoss, Oda Astrid Haarr
dc.date.accessioned2015-10-06T07:34:36Z
dc.date.available2015-10-06T07:34:36Z
dc.date.created2015-06-25
dc.date.issued2015
dc.identifierntnudaim:10598
dc.identifier.urihttp://hdl.handle.net/11250/2351637
dc.description.abstractLung cancer is the number one cause of worldwide cancer deaths for men, and second cause of cancer deaths for women. Tobacco smoking is strongly associated with lung cancer, and carcinogenic compounds inhaled when smoking cause DNA damage of lung tissue when they are metabolized in the cells. Epithelial-mesenchymal transition (EMT) is a process in tumorigenesis where the cells gain an invasive and migratory phenotype, and it mediates metastasis of the primary tumor. EMT is characterized by specific markers expressed from the mesenchymal cells. A possible connection between steroid receptor pathways and carcinogens has been proposed, and transcription factors (FOXA family) regulating these pathways may therefore play an important part in regulating carcinogen metabolism. FOXA1 and FOXA2 are downregulated in human bronchial epithelial cells (HBECs) that are exposed to cigarette smoke condensate. As these cells also have gained a mesenchymal phenotype, this downregulation supports the notion that FOXA factors may be involved in EMT. Ectopic expression of FOXA1 in the transformed HBECs is an interesting way of studying whether the factor alone plays a significant part in EMT. In this thesis, we aimed to 1) Develop a method for controlled integration of FOXA1 into transformed HBEC cells 2) Restoring close to normal expression levels in order to study the role of FOXA1 in regulating mesenchymal markers and phenotype To achieve these goals, we used CRISPR-Cas9 system for RNA guided site-specific genome engineering, and designed an expression construct to be integrated into the target genome at a genomic safe harbor target locus. The CRISPR-Cas9 system creates a double strand break at a desired locus, and this break may be repaired by homologous recombination using a repair construct containing the gene of interest (FOXA1). The FOXA1 construct was chemically synthesized as three gene fragments (gBlocks) of ~2000 bp to be assembled using the Gibson Assembly method. After assembly, the construct would be inserted into the AAVS1 safe harbor locus, a site widely used for gene knock-in with no adverse effects reported. In the course of this thesis, assembly of the gBlocks into a FOXA1 construct could not be achieved, and this was probably caused by the overlapping regions needed for Gibson Assembly being too long, and by unspecific products in the PCR-amplified gBlocks inhibiting the reaction. The two custom guide RNA-sequences required for the CRISPR-mediated double strand break were designed, and one of them (gRNA2) was successfully cloned into a CRISPR delivery plasmid vector. The remaining steps required to investigate the role of FOXA1 in EMT is to redesign and successfully assemble the FOXA1 construct, clone gRNA1 into a CRISPR delivery vector, and co-transfect HBEC cells with the FOXA1 construct and guide RNA-containing CRISPR vectors so FOXA1 can be expressed in the cells. Once established, the in vitro site-specific knock-in system will provide a versatile tool for future knock-in studies in HBEC cells and other human in vitro models.
dc.languageeng
dc.publisherNTNU
dc.subjectBioteknologi
dc.titleRole of FOXA genes in maintaining transformed phenotype in human bronchial epithelial cells - Construction of a CRISPR-Cas9 vector system for site-specific integration of FOXA1
dc.typeMaster thesis
dc.source.pagenumber114


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