Fare Miyoblast Hücre Hattinda (C2c12 Hücre Hatti) Klf5 Geni Üzerinde Hedefli İnsersiyon Gerçekleştirilmesi

Abstract

Klf5 is a zinc finger transcription factor that is expressed in early embryonic stem cells as well as adult somatic epithelial tissue. The function of Klf5 is diverging in a context dependent manner in cells and tissues. During development, Klf5 has a role in the maintenance of undifferentiated state in embryonic stem cells. Moreover, Klf5 is also acting on cellular processes such as cell migration, apoptosis, inflammation, angiogenesis and differentiation. Previous studies showed a novel role for Klf5 as a regulator of proliferation and differentiation in skeletal muscle stem cells. Detection of Klf5 at the protein level harbor technical obstacles. Commercially available antibodies exhibit low affinity, low specificity and fail to recognize post-translationally modified forms that is directly relevant to the function. Since these obstacles prevent further functional protein studies such as western blots, protein co-immunoprecipitation and chromatin immunoprecipitation (ChIP) assays, genome editing applications such as CRISPR/Cas9 system provide solution opportunities. This thesis is conceptuated to establish a stable cell line that express a V5-epitope tag within the N-terminal of Klf5 protein. Targetted insertion via CRISPR/Cas9 system is suggested to overcome the above mentioned obstacles. An N-terminal V5 epitope tag would not interfere with the Klf5 function and enable the use of an anti-V5 antibody in the established C2C12 mouse myoblast cells to identify endogeneous Klf5. This tool will aid in for the advancement of the current functional protein studies.