BLT Stages

Abstract Bachelor Project 2019-2020: Generation of stable CRISPR-cas9-expressing cell lines and knock-out mutants for studies of epithelial mesenchymal transition in osteosarcoma

Osteosarcoma (OS) is a type of bone cancer that is primary malignant. It mostly occurs in teenagers and young adults, but it can also occur in younger children and older adults. The therapeutic options for treating metastatic disease is limited, it is important to find new treatments to improve the survival rate of metastatic patients. The aim of this study is to generate mutant cancer lines using the CRISPR-Cas9 technology, the target genes will be the transcription factors Sp1 and p53, using the osteosarcoma cell line U20S. The four main goals of this study are:

• Establish cell stocks and routine cultures of U20S and H-cat cells.
• Generate p53 and Sp1 knock-out mutants in U20S cell lines using CRISPR-Cas9/sgRNA for p53 and Sp1.
• Analyze protein and mRNA expression of p53 and Sp1 in knock-out mutants.
• Analyze cell viability and apoptosis induction of knock-out mutants treated with Turmeric.

Generation of mutant cancer cells by using CRISPR-Cas9 technology. Then, the knock-out of the transcription factors Sp1 and p53 were analyzed by different methods. The protein expression is controlled via western blot, mRNA through reverse transcription-Polymerase Chain Reaction (RT-PCR). The cell viability via MTT and apoptosis were verified after the knock-out mutants were treaded with Turmeric. Many different EMT genes are regulated by transcription factor (TF) Sp1. This results in up- or down regulation of EMT markers; E- and N-cadherin, Vimentin, Snail. Sp1 has also an effect on cell proliferation, invasion and adhesion. Many initial studies have been conducted to identify certain genes that have a potential to serve as treatment, but further research is necessary.