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• W1786618410 abstract "ABSTRACTTelomerase activity is a potential molecular marker for cancer and primitive cells in regenerative tissues. The most commonly used method to measure telomerase activity in cells is the telomerase reverse amplification protocol (TRAP) assay, where the protein extracts derived from homogenized cells are analysed. In this thesis we focused on developing a method where telomerase activity could be monitored and used to separating cells with high telomerase activity without killing them. We used the promoter sequence of human telomerase reverse transcriptase (hTERT) for transcriptionally controlling a destabilised green fluorescence protein (d2EGFP) reporter gene since there is a strong correlation between hTERT transcriptional activity and telomerase activity in cells. Moreover, we chose to work with adenoviral vectors due to their high level of transient gene expression as a gene transfer system. Unfavourably, adenoviral vector transduction of tumour cells has been hampered by low expression of the coxsackie B and adenovirus receptor (CAR) on the host cells, which is the attachment receptor for the most commonly used adenoviral vector (Ad5). Thus, we used a fibre-modified Ad5 vector with the tropism of Ad35, which uses the ubiquitously expressed CD46 protein as attachment receptor. We transduced a variety of cell lines with the developed hTERT-d2EGFP vector and demonstrated a close correlation between expression levels of the d2EGFP, endogenous telomerase activity, and hTERT mRNA expression. Furthermore, the hTERT-d2EGFP reporter vector was able to monitor the cell cycle and differentiation stage-dependent hTERT transcription activity.Additionally, in order to study the self-renewal process of haematopoietic stem cells (HSCs), we utilised the hTERT-reporter vector for monitoring hTERT transcription activity in human HSCs. Interestingly, increasing hTERT expression was inversely correlated to the self-renewal capacity among primitive HSCs and the hTERT-reporter vector could be used to separate short-term from long-term re-populating human HSCs. In summary, we have developed a versatile adenoviral vector system with Ad35 tropism, which can be used to transiently transduce tumour cells as well as haematopoietic cells. This vector system can be used to study normal regulation of hTERT and has potential in development of novel therapeutic approaches for treatment of tumours and malignancies in the blood system." @default.
• W1786618410 created "2016-06-24" @default.
• W1786618410 creator A5000691373 @default.
• W1786618410 date "2007-01-01" @default.
• W1786618410 modified "2023-09-28" @default.
• W1786618410 title "Development of adenoviral vectors for monitoring telomerase activity in living cells" @default.
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