The CRISPR-Cas system derived from bacteria and archaea adaptive immune system is a high potential method for fast genome editing that promises to revolutionize previous genome engineering. It is based on the specific targeted induced double strand break by an endonuclease. In elapsed studies PA28γ figured out as an important key molecule involved in cell cycle regulation, cell signaling transcription, immune response and apoptosis. Recent investigations showed p53 to be a target of PA28γ enhanced ubiquitination via MDM2 and subsequent proteasomal degradation. Otherwise mutant p53 (R248Q) has been shown as suppressor of the REGγ promotor. This study aimed the CRISPR-Cas mediated gene knockout of PSME3 and tp53 in Caspase3 lacking MCF-7 breast cancer cells to investigate apoptosis.
A user-developed protocol was established to implement the Multiplex CRISPR/Cas9 Assembly System Kit and the alone standing pSpCas9(BB)-2A-GFP plasmid provided by Takashi Yamamoto and Feng Zhang (Ran et al. 2013, Sakuma et al. 2014) for the generation of knockout cells.
The cloning of gRNA harboring plasmids targeting PSME3 exon1/exon4 as well as tp53 exon1_1/exon1_2 was fast and in a high efficient fashion but a verification of the final constructs via T7 Endonuclease I assay was not possible.
Interestingly, using fluorescent microscopy different gRNAs cloned in the CRISPR plasmids revealed variant apparent transfection efficiencies or GFP plasmid or protein stability. Furthermore, PA28γ targeted cells showed a better survival than p53 knockout cells. Therefore, also no tp53 targeted cells survived the serial dilution and clonal selection over an eight week period. PSME3 exon1_F1, exon4_C8 and exon4_B9 revealed PA28γ levels of about 50% compared to the untransfected wild type cells in Western Blot analyses. This could be caused by a heterozygous knockout of the PSME3 gene on chromosome17. One single cell clone (PSME3 exon4_F9) maybe carrying a gain of the PSME3 gene, undergoing interchromosomal recombination or only was hidden at one allele by the Cas9 enzyme showed 75% for PA28γ levels.
In summary CRISPR-Cas enabled us probable to modify the PSME3 and tp53 gene in MCF-7 cells resulting in altered survivals of the transfected cells. Additionally, first investigations of the new established MCF-7 PSME3 knockout cell lines considering the PA28γ protein level showed a successful 50% reduction. It was not possible to study any apoptosis related behavior.
Inhaltsverzeichnis (Table of Contents)
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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
- 2.1 Materials
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.
- Quote paper
- Marcel Schlecht (Author), 2016, Functional analysis of PA28γ in MCF-7 breast cancer cells by overexpression & CRISPR-Cas9 mediated gene silencing, Munich, GRIN Verlag, https://www.grin.com/document/366894
