Green and red fluorescent protein tagging of endogenous proteins in glioblastoma using the CRISPR (clustered regularly interspaced short palindromic repeats)-Cas9 system

Glioblastoma multiforme is the most malignant primary brain tumor that affects adults, recognized by the World Health Organization as an aggressive grade IV astrocytoma. Patients diagnosed with this type of tumor are left with a poor prognosis even with the most advanced treatment available. The cancer is quite heterogeneous and is typically categorized into four different subtypes depending on genetic aberrations and patient characteristics. Furthermore, researchers have discovered a subpopulation of glioblastoma cells, known as cancer stem cells, which are thought to be resistant to current therapies and responsible for tumor reoccurrence and relapse. Previous studies, in addition to this one, have found that the differentiation of glioblastoma cells downregulate nestin protein expression, the selected stem cell marker, and upregulate glial fibrillary acid protein expression, the selected differentiation marker, using immunofluorescence. Thus, one alternative treatment option is to understand the mechanism underlying the differentiation of cancer stem cells. Four cell cultures representative of each glioblastoma subtype will be endogenously tagged using the genome editing system, Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR/Cas9). The representative stem cell marker, nestin, will be tagged with a green fluorescent protein, while the chosen differentiation marker, glial fibrillary acid protein, will be tagged with a red fluorescent protein. Several drugs were screened to analyze whether the drugs had a differentiation effect on the glioblastoma cells. As a result, strong evidence indicated that bone morphogenetic protein four upregulated glial fibrillary acid protein expression levels to the same extent as the differentiation control media using 5% fetal bovine serum. The goal of this study is to establish a method to directly monitor the differentiation process of glioblastoma cells as a novel molecular screening method. In this case, all glioblastoma cells, even the ones resistant to treatment, can be eliminated through an initial “pre-treatment” by forcing differentiation of cancer stem cells, making the cells more susceptible to the chemotherapy drugs. In the long run, glioblastoma patients would have a chance at a more positive prognosis; a longer life that is free of glioblastoma.Master Thesis in Applied Biotechnolog