• Maja Balling Zacher Sørensen
4. semester, Medicin med Industriel Specialisering, Kandidat (Kandidatuddannelse)
Leukemic stem cells (LSCs) are mutated hematopoietic stem or progenitor cells that can initiate acute myeloid leukemia (AML). LSCs are a source of relapse, as they are often more resistant to conventional chemotherapies and therefore, the identification of drugs that effectively target LSCs is of great importance for establishing more effective treatments towards AML. However, there is no simple way to identify the LSCs since the immunophenotype varies from case to case. The ERG+85 enhancer promoter relates to the endogenous ERG expression which correlates to stemness state. A reporter system based on this has recently been shown potential for the identification of cellular stemness. The overall objective in the present thesis was to implement a novel method for identifying LSCs using a lentiviral stemness reporter system, which could allow exploring compounds that target LSCs in AML. In order to achieve this, a lentivirus was produced containing the vector pMIN-ERG+85. The vector contains a constitutive green fluorescent protein (GFP) reporter and a blue fluorescent protein (BFP) gene under the ERG+85 enhancer promoter. Plasmids for lentiviral production was transformed into competent Escherichia coli XL1-blue cells with either pMIN-ERG+85 or the control vector pMIN. HEK 293T cells were then transfected with the plasmids to produce lentivirus, either containing pMIN-ERG+85 or pMIN. The AML cell lines Kasumi-1, KG-1, and MOLM-13 were transduced with the lentiviral vectors. Infected cells were sorted by FACS and long term cultured, in order to expand the number of cells for drug screenings. Infected cells were repeatedly assessed by flow cytometry. Results showed that transduction was enhanced by the use of polybrene transduction reagent, that lentiviral vectors were able to infect Kasumi-1 and MOLM-13 but not KG-1, and that transduction were more efficient with pMIN than pMIN-ERG+85. Moreover, showed infected cells a higher BFP signal in the control vector, pMIN, compared to pMIN-ERG+85 vector suggesting leaky promoter activity of the mCMV promoter. The drug screen assessments could not be implemented due to time restrictions, and it can be concluded that further investigations are needed regarding the higher BFP signal in the control vector.
SprogEngelsk
Udgivelsesdato5 aug. 2020
Antal sider55
Ekstern samarbejdspartnerBRIC, University of Copenhagen
Professor Krister Wennerberg krister.wennerberg@bric.ku.dk
Praktiksted
ID: 338221788