Regulation of gene expression driven by endogenous and viral promoters in Chinese Hamster Ovary cells using Triplex-Forming OligonucleotidesPapež, Maja (Avtor) Narat, Mojca (Mentor) Baumann, Martina (Komentor) recombinantgene expressiontriplex forming oligonucleotidesCHO cellspromoterβ-ActinRAD52HSP90ST6GAL1Industrial production of recombinant proteins in CHO cells commonly uses vectors possessing strong viral promoters. Though this results in high productivity, it is accompanied by cellular stress and economic loss. In this thesis, the viral CMV and four endogenous CHO promoters (β-Actin, RAD52, HSP90, ST6GAL1) driving different reporter genes were used. Several TFOs were designed for each promoter. TFOs can affect expression of a reporter gene by binding into the promoter region. The activity of each promoter was observed with the luciferase assay and later with flow cytometry, when a different reporter gene was used. The next step included the use of TFOs, first preincubated with the promoter containing plasmid pre-transfection, then interacting with the promoter by being endogenously transcribed from a separate plasmid. We have also observed the TFO’s effect on an endogenous ST6GAL1 promoter in two CHO cell lines and monitored triplex formation by using FRET-qPCR. Some TFOs decreased promoter activity, which was an expected form of action. Yet in some cases, an increase in promoter activity was observed. The molecular structure of the TFOs and their sequence-specific placement in the promoter enables interaction with different TFs, prevention of PIC assembly and even altering the chromatin state. If the role of the TFOs would be elucidated and predictable, they could become an important metabolic engineering switch in recombinant protein production.[M. Papež]20202020-05-23 07:15:04Magistrsko delo/naloga116212UDK: 601.4:577.21:602.6:587/579:606:62(043.2)VisID: 175817COBISS_ID: 18310147sl