| Abstract [eng] |
Pancreatic ductal adenocarcinoma (PDAC) is a highly lethal malignancy characterized by a severely hypoxic tumor microenvironment (TME), which promotes tumor progression and therapy resistance. Hypoxia-inducible factors (HIFs) are central mediators of the cellular response to low- oxygen conditions, but their isoform-specific functions in PDAC remain insufficiently understood. This thesis aimed to establish the Multiple Lentiviral Expression (MuLE) system as a platform for the isoform-specific knockout of HIF1A, HIF2A, and HIF3A in the human PDAC cell line MIA PaCa-2. The objectives included the construction of single-guide RNA (sgRNA) and Cas9-expressing MuLE vectors, generation of lentiviral particles, optimization of transduction and selection protocols, and preliminary transcriptional profiling of wild-type cells under hypoxia. Functional MuLE constructs were successfully assembled and validated. Transduction and selection of MIA PaCa-2 cells were optimized using fluorescence and antibiotic resistance. In parallel, wild-type cells were subjected to defined hypoxic conditions, with and without pharmacological inhibition of HIF-1α using the small-molecule inhibitor CAY10585. Transcriptional profiling revealed a general downregulation of HIFs and selected hypoxia target genes under hypoxia, whereas HIF-1α inhibition partially restored their expression, suggesting a complex, context-dependent regulatory mechanism. Together, this work demonstrates the potential of the MuLE system for isoform-specific gene targeting in PDAC and provides a methodological foundation for further functional studies. Continued work will be required to validate knockout efficiency and characterize isoform-specific phenotypes to investigate the distinct roles of HIF isoforms in the cellular adaptation to hypoxia. |
