Versuche zur Etablierung von CrisprCas9 in chondrogenen Progenitorzellen

by Evi Tsiakourma
Doctoral thesis
Date of Examination:2024-10-02
Date of issue:2024-09-17
Advisor:Prof.Dr. Nicolai Miosge
Referee:Prof. Dr. Philipp Kaufmann
Referee:Prof. Dr. Thomas Meyer
Referee:Prof. Dr. Margarete Schön
Persistent Address: http://dx.doi.org/10.53846/goediss-10725

 

 

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Abstract

English

Osteoarthritis is the most common form of joint disease and is therefore one of the main causes of mobility limitations in older age. Current treatment models do not yet include curative therapies. The “Oral Biology and Tissue Regeneration” research group is intensively intrested in developing a cell-based therapy for osteoarthritis, focusing on chondrogenic progenitor cells. Chondrogenic progenitor cells are a subpopulation of chondrocytes that have been isolated from patients with advanced osteoarthritis and identified as stem cells. Understanding and advancing cell-based cartilage regeneration using stem cells could lead to a promising therapy. The CRISPR-Cas9 system is a method for targeted gene modification, allowing genes to be inserted, removed, or altered. In prokaryotes, it serves as an adaptive defense mechanism to protect against viral genetic material. In recent decades, this natural defense mechanism has been identified as a universal research tool, enabling genome editing. In this work, strategies were tested to optimally establish the CRISPR-Cas9 method in chondrogenic progenitor cells. Using this technology, the chondrogenic potential of chondrogenic progenitor cells should be enhanced through the targeted knockout of specific genes. During the establishment phase, the spCas9 plasmid was transfected based on a dual gRNA strategy. The target genes RAN and LEMD2 were selected as potential interaction partners of the osteogenic transcription factor Runx2, identified in a previous study. Following the successful establishment of the transfection, sorting was performed using flow cytometry. Additionally, conditions for successful subcultivation and isolation of clones were investigated. Based on this work, essential conclusions for the successful establishment of the method in chondrogenic progenitor cells were drawn, so that in subsequent projects, clones could hopefully be successfully isolated and studied using this technology
Keywords: CPC; Crispr-Cas9; Osteoarthritis