Regulation of gene expression driven by endogenous and viral promoters in Chinese Hamster Ovary cells using Triplex-Forming Oligonucleotides

Papež, Maja (author), Narat, Mojca (mentor), Baumann, Martina (comentor)

Industrial 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.

• Language: English
• Keywords: recombinant, gene expression, triplex forming oligonucleotides, CHO cells, promoter, β-Actin, RAD52, HSP90, ST6GAL1
• Work type: Master's thesis/paper
• Typology: 2.09 - Master's Thesis
• Organization: BF - Biotechnical Faculty
• Publisher: [M. Papež]
• Year: 2020
• PID: 20.500.12556/RUL-116212
• UDC: 601.4:577.21:602.6:587/579:606:62(043.2)
• COBISS.SI-ID: 18310147
• Publication date in RUL: 23.05.2020

Secondary language

• Language: Slovenian
• Title: Regulacija vodenja izražanja genov z endogenimi in virusnimi promotorji z uporabo tripleks snujočih oligonukleotidov

Abstract

Industrijska proizvodnja rekombinantnih proteinov v celicah CHO pogosto posega po vektorjih z močnimi virusnimi promotorji. Njihova uporaba se odraža v visoki produktivnosti, ki pa jo hkrati spremlja celični stres in ekonomske izgube. V tej nalogi smo uporabili virusni promotor CMV ter štiri endogene promotorje CHO (β-aktin, RAD52, HSP90, ST6GAL1), ki so vodili različne poročevalske gene. Za vsakega izmed promotorjev smo načrtovali TFO, ki lahko prek vezave v promotor uravnavajo prepisovanje poročevalskega gena. Aktivnost promotorjev smo opazovali s pomočjo luciferaznega testa. Ko smo uporabili drug poročevalski gen, pa še s pretočno citometrijo. Naslednji korak je bil uporaba posameznega TFO, predinkubiranega s plazmidom, ki je vseboval posamezen promotor. Kasneje smo celice CHO kotransfecirali s plazmidom, ki je vseboval posamezen promotor ter hkrati s plazmidom, ki je vseboval zapis za posamezen TFO. Preizkusili smo tudi odziv endogenega promotorja ST6GAL1 v dveh celičnih linijah CHO ter spremljali tvorbo tripleksa s pomočjo FRET-qPCR. Nekateri TFO so aktivnost promotorja znižali, kar je bilo pričakovano. Presenetljivo pa se je v nekaterih primerih izražanje gena povišalo. Struktura TFO in njihova zaporedno-specifična vezava v promotor omogoča interakcijo TFO z različnimi TF. TFO lahko preprečijo tvorbo PIC in celo spremenijo stanje kromatina. Če bi bila njihova vloga jasna in predvidljiva, bi lahko postali pomembno metabolno stikalo v proizvodnji rekombinantnih proteinov.

• Keywords: rekombinanten, izražanje genov, tripleks snujoči oligonukleotidi, celice CHO, promotor, β-aktin, RAD52, HSP90, ST6GAL1