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

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 CRIRSPR-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 (PA28γ)

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

2.1.9 Plasmids, oligonucleotides, primer

2.1.10 Private computer software

2.1.11 Buffers and solutions

2.2 Methods

2.2.1 Cell biological methods

2.2.2 Molecular biological methods

2.2.3 Biochemical methods

2.2.4 General methods

3. RESULTS

3.1 In silico design, cloning and characterization of gRNA harboring plasmids in transfected MCF-7 cells

3.1.1 In silico analyses of genomic regions related to PSME3 exon1, exon4 and tp53 exon1_1/2 for gRNA design

3.1.2 gRNA encoding oligo nucleotides were cloned into pX330A-1x2 and pX458 plasmids

3.1.3 Apparent transfection efficiencies vary with gRNA expressing constructs

3.1.4 Different gRNA constructs reveled a various outcome in MCF-7 cells

3.2 Confirmation of gRNA constructs effects on a genomic level

3.2.1 Establishment of the genomic PCR for PSME3 and tp53 gene

3.2.2 Verification of gRNA targeting efficiencies using the T7 Endonuclease I assay

3.3 Transfection of MCF-7 cells with gRNA harboring pX330A1x2 and pX458 plasmids

3.3.1 Survival rates of single cell clones are linked to the gRNA construct

3.3.2 MCF-7 cells transfected with pX330A-1x2_PSME3exon1 and pX330A-1x2_PSME3exon4 showed a heterozygous knockout of the PSME3 gene

3.3.3 The PSME3 exon4_F9 clone revealed an anomalous PA28γ level

4. DISCUSSION

4.1 The CRSIPR-Cas9 system seems to be an innovative method for fast genome engineering but it is not that easy to use as described in literature

4.2 The transfection of MCF-7 cells with different gRNA constructs revealed variant survival

4.3 For fast generation of mutant cell lines using CRISPR-Cas9 automated monitoring is necessary

4.4 Generated knockout effects of PA28y in MCF-7 cells

5. OUTLOOK

6. SUPPLEMENT

6.1 Plasmid chart pX330A-1x2

6.2 Pasmid chart pX330S-2

6.3 Plasmid chart pX458

6.4 CRISPR-Cas transfected cell Western Blot verfiifcation

6.5 Poster Abstract VideoScan Applications in CRISPR-Cas9 Monitoring

7. REFERENCES

Research Objectives and Core Themes

This thesis investigates the application of the CRISPR-Cas9 genome editing system for the targeted knockout of the PSME3 (encoding PA28γ) and tp53 genes in the Caspase-3 deficient MCF-7 breast cancer cell line, specifically aiming to characterize the subsequent cellular survival and potential apoptotic changes.

• Functional role of PA28γ in protein degradation and apoptosis.
• Implementation of CRISPR-Cas9 cloning and transfection protocols.
• Analysis of gRNA efficiency and genomic knockout verification.
• Evaluation of phenotypic outcomes and survival in edited MCF-7 cell clones.

Excerpt from the Book

Summary of Chapters

1. INTRODUCTION: Provides a background on genome engineering history, the microbial origins of CRISPR-Cas9, the experimental design for gRNA engineering, and the specific cell and protein models used in the study.

2. MATERIAL AND METHODS: Details the materials, equipment, cell culture conditions, transfection protocols, cloning strategies, and biochemical methods, including DNA isolation, PCR, and Western Blotting.

3. RESULTS: Presents the findings regarding gRNA design, cloning success, transfection efficiency, survival rates of cell clones, and the verification of PA28γ knockout via Western Blot analysis.

4. DISCUSSION: Analyzes the experimental success, limitations regarding transfection and genomic verification, the impact of gRNA constructs on survival, and the implications of cellular heterogeneity.

5. OUTLOOK: Suggests future directions for establishing more robust PCR and screening methods, improving clonal selection, and further investigating apoptotic pathways in PA28γ knockout cells.

6. SUPPLEMENT: Includes detailed plasmid maps, additional Western Blot validation data, and a conference abstract related to the VideoScan monitoring technology.

7. REFERENCES: Lists the cited scientific literature supporting the methodologies and theoretical frameworks applied in the study.

Keywords

CRISPR-Cas9, PA28γ, PSME3, MCF-7, Breast Cancer, Gene Knockout, Genome Engineering, Apoptosis, p53, Western Blot, Transfection, gRNA, Clonal Selection, VideoScan, Molecular Biology

Frequently Asked Questions

What is the core focus of this Master's thesis?

The thesis focuses on the functional analysis of the PA28γ protein in MCF-7 breast cancer cells by utilizing the CRISPR-Cas9 genome editing system to perform gene knockouts and evaluate the biological consequences.

Which genes were targeted for knockout?

The study specifically targeted the PSME3 gene (which encodes PA28γ) and the tp53 gene in MCF-7 cells.

What is the primary objective of this research?

The primary goal was to establish the CRISPR-Cas9 laboratory workflow and investigate the effects of targeted gene disruption on the transfected MCF-7 cell survival and apoptotic behavior.

What scientific methods were employed?

Key methods included molecular cloning of gRNA-harboring plasmids, Lipofectamine-mediated transfection, fluorescence-based clonal selection, genomic PCR, T7 Endonuclease I assays, and Western Blot analysis for protein quantification.

What are the main topics discussed in the main body?

The main body covers the in silico gRNA design, the methodology for constructing CRISPR plasmids, the transfection process, the confirmation of genomic effects, and the statistical analysis of cell survival and protein expression levels.

Which keywords best describe this research?

CRISPR-Cas9, PA28γ, MCF-7, Breast Cancer, Gene Knockout, and Genome Engineering are among the most descriptive keywords for this study.

Why was the MCF-7 cell line chosen for these experiments?

MCF-7 is a well-established breast cancer cell line that allows researchers to study the impacts of PA28γ and p53, noting specifically that these cells are known to be Caspase-3 deficient.

What were the main conclusions regarding transfection efficiency?

The research found that transfection efficiency is heavily influenced by the specific gRNA construct used, with PSME3-targeting constructs displaying lower apparent efficiencies compared to tp53-targeting ones, often correlated with cell viability issues.

What was the outcome of the PA28γ knockout?

Western Blot analyses confirmed a successful reduction in PA28γ protein levels by approximately 50%, which was interpreted as a stable heterozygous knockout in the tested clones.