Functional analysis of PA28γ in MCF-7 breast cancer cells by overexpression & CRISPR-Cas9 mediated gene silencing

Inhaltsverzeichnis (Table of Contents)

• 1. INTRODUCTION
  • 1.1 CRISPR-Cas9 System
  • 1.2 The microbial origin of CRISPR-Cas9
    • 1.2.1 Components of the adaptive immune system in streptococcus pyrogenes (s. pyrogenes)
    • 1.2.2 Mechanism of adaptive immune response in s. pyrogenes
  • 1.3 Experimental design for precise CRISPR-Cas9 mediated gene engineering
    • 1.3.1 Guide RNA (gRNA) design
    • 1.3.2 Construction of gRNA harboring plasmids
    • 1.3.3 Confirmation of gRNA function
    • 1.3.4 Advantages and limitations of CRISPR-Cas
  • 1.4 Michigan Cancer Foundation 7 (MCF-7) breast cancer cell line
  • 1.5 Proteasome activator 28 gamma (PA28y)
  • 1.6 Preliminary work and aim of the study
    • 1.6.1 Preliminary work and background of the study
    • 1.6.2 Aim of the study
• 2 MATERIAL AND METHODS
  • 2.1 Materials
    • 2.1.1 Laboratory equipment and instruments
    • 2.1.2 Plastic materials and consumables
    • 2.1.3 Chemicals, buffers and media for cell biology
    • 2.1.4 Chemicals, buffers and enzymes for molecular biology
    • 2.1.5 Chemicals and materials for biochemistry
    • 2.1.6 Cell lines and bacteria
    • 2.1.7 Antibodies
    • 2.1.8 Kits

Zielsetzung und Themenschwerpunkte (Objectives and Key Themes)

This master thesis focuses on investigating the impact of CRISPR-Cas9 mediated gene silencing on apoptosis in MCF-7 breast cancer cells. It specifically explores the role of PA28y, a key molecule involved in various cellular processes including cell cycle regulation, immune response, and apoptosis, in relation to the p53 gene. The study aims to elucidate the effects of PA28y knockout on cell survival and potential apoptosis-related behavior in these cancer cells.

• CRISPR-Cas9 mediated gene editing
• Apoptosis in breast cancer cells
• PA28y's role in cell cycle regulation, immune response, and apoptosis
• The interaction of PA28y and p53
• Investigating the effects of PSME3 and tp53 gene knockout on cell survival and apoptosis in MCF-7 cells

Zusammenfassung der Kapitel (Chapter Summaries)

The first chapter provides a comprehensive introduction to the CRISPR-Cas9 system, its origins, and its applications in gene engineering. It delves into the components of the adaptive immune system in bacteria, particularly in Streptococcus pyogenes, and explains the mechanism of the CRISPR-Cas9 system in targeting and modifying specific genes. The chapter also outlines the experimental design employed for precise CRISPR-Cas9 mediated gene editing, emphasizing key aspects like gRNA design, construction of gRNA harboring plasmids, and the validation of gRNA function. Furthermore, it highlights the advantages and limitations of CRISPR-Cas9 technology. The chapter concludes with an introduction to MCF-7 breast cancer cells and the role of PA28y in cellular processes.

Chapter 2 details the materials and methods employed in the study. It outlines the equipment, consumables, chemicals, buffers, enzymes, cell lines, bacteria, antibodies, and kits used in the experiments.

Schlüsselwörter (Keywords)

The core concepts explored in this thesis are CRISPR-Cas9, gene editing, apoptosis, breast cancer cells, MCF-7 cell line, PA28y, PSME3, tp53, MDM2, ubiquitination, proteasomal degradation, and cell survival.