In this master thesis, we want to develop and implement simultaneous genome and transcriptome analysis at the single cell level, through analyzing single cells of human embryonic stem cells and human fibroblasts. For the validation of the experiments we used (1) purified RNA & DNA mixes, (2) larger numbers of cells and (3) dilutions down to one single cell.
The ultimate goal of using such technique is to investigate each single blastomere of human embryos at the two- to eight-cell stage. We particularly want to investigate the hypothesis whether shortage of previously mentioned transcripts leads to an abnormal number of chromosomes during embryonal development.
Roughly half of the human preimplantation embryos obtained after in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI) show a high frequency of chromosomal mosaicism, the event where not all cells of an embryo display an identical chromosomal composition. These abnormalities are mostly explained by anaphase lagging, non-disjunction and endoreduplication, of which anaphase lagging is the most common mechanism resulting in mosaicism.
They originate during postzygotic, mitotic divisions. Several authors hypothesize that the causes of the aberrations lie in the depletion of certain transcripts such as for the components of the chromosomal passenger complex (CPC) and the spindle attachment checkpoint (SAC) which are key regulators during cell division that correct chromosome attachment errors and prevent chromosome mis-segregations and aneuploidy.
Conversely, transcriptome analysis on whole embryos has shown the presence of these transcripts which argues against their role during the development of aneuploidies. Interestingly, different SAC and CPC expression levels were shown at the single cell level, but without correlation to chromosomal abnormalities.
Table of Contents
1. INTRODUCTION
1.1 ASSISTED REPRODUCTIVE TECHNOLOGIES
1.2 PREIMPLANTATION GENETIC TESTING
1.2.1 PGT FOR MONOGENIC/SINGLE GENE DEFECT (PGT-M)
1.2.2 PGT FOR ANEUPLOIDIES (PGT-A)/STRUCTURAL REARRANGEMENTS (PGT-SR)
1.2.2.1 FLUORESCENCE IN SITU HYBRIDIZATION FOR SINGLE CELL ANALYSIS
1.2.2.2 COMPREHENSIVE CHROMOSOME SCREENING
1.2.2.3 NEXT GENERATION SEQUENCING
1.3 THE ORIGIN OF CHROMOSOMAL ABNORMALITIES IN PREIMPLANTATION EMBRYOS
1.3.1 ORIGIN OF MITOTIC ANEUPLOIDIES
1.3.1.1 UNDERLYING PROCESSES LEADING TO ABNORMAL CHROMOSOME NUMBERS
1.3.1.2 MATERNAL FACTORS AFFECTING MITOTIC ANEUPLOIDIES
1.3.1.3 THE SPINDLE ASSEMBLY CHECKPOINT DURING MITOSIS
1.3.1.4 THE CHROMOSOMAL PASSENGER COMPLEX
2. OBJECTIVES OF THE MASTER THESIS
3. MATERIALS AND METHODS
3.1 CELL CULTURE
3.2 RNA/DNA EXTRACTION
3.3 SINGLE CELL PICK-UP
3.4 CDNA SYNTHESIS
3.5 CDNA AMPLIFICATION
3.6 PHYSICAL SEPARATION OF MRNA AND GDNA
3.7 MEASURING CDNA CONCENTRATIONS
3.8 GENOMIC DNA PRECIPITATION AND AMPLIFICATION
3.9 QUANTITATIVE REAL-TIME PCR (QRT-PCR)
3.10 PCR
4. RESULTS
4.1 VALIDATION OF THE OLIGO-DT 30VN PRIMER SET
4.1.1 BULK RNA FIBROBLASTS
4.1.2 BULK RNA HESC
4.2 VALIDATION OF THE TSO AND ISPCR PRIMER SET
4.3 IMPLEMENTATION OF OLIGO-DT 30VN LABELLED MAGNETIC BEADS
4.3.1 RNA DILUTION SERIES
4.3.2 SINGLE CELL LEVEL
4.4 VALIDATION OF THE CFTR PRIMER SET
4.4.1 DNA DILUTION
4.4.2 SINGLE CELL LEVEL
4.5 VALIDATION OF PHYSICAL SEPARATION OF MRNA AND GDNA
4.5.1 RNA/DNA MIXES
4.5.1.1 VALIDATION OF AMPLIFIED CDNA
4.5.1.2 VALIDATION OF AMPLIFIED GDNA AFTER MULTIPLE DISPLACEMENT AMPLIFICATION
4.5.2 SINGLE CELL LEVEL
4.5.2.1 VALIDATION OF AMPLIFIED CDNA
4.5.2.2 VALIDATION OF AMPLIFIED GDNA AFTER MULTIPLE DISPLACEMENT AMPLIFICATION
4.5.2.3 VALIDATION OF THE PICOPLEX WGA KIT
4.5.2.4 VALIDATION OF AMPLIFIED GDNA AFTER PICOPLEX
4.5.2.5 SHALLOW WHOLE GENOME SEQUENCING DATA
5. DISCUSSION
6. CONCLUSION
Research Objectives and Themes
This master thesis aims to develop and implement a protocol for simultaneous genome and transcriptome analysis at the single-cell level, specifically using human embryonic stem cells and fibroblasts, to investigate whether a deficiency in specific transcripts contributes to the development of chromosomal abnormalities during embryogenesis.
- Development of a simultaneous genomic and transcriptomic analysis pipeline.
- Validation of physical mRNA and genomic DNA separation techniques.
- Evaluation of cDNA and genomic DNA amplification methods at the single-cell level.
- Investigation into chromosomal mosaicism and transcript expression profiles.
- Application of shallow whole genome sequencing to detect chromosomal aberrations.
Excerpt from the Book
1.3.1.3 The spindle assembly checkpoint during mitosis
The SAC is a mitotic feedback-control system that prevents onset of anaphase and chromosome segregation, until all chromosomes are properly attached to the mitotic spindle (78). Thus, it avoids the development of aneuploidies by arresting the cell in mitosis and blocking transition into the ultimate steps of cell division (79). It consists of several proteins such as the sensor protein mitotic arrest deficient 1 (MAD1), budding uninhibited by benzimidazoles 1 (BUB1), and multipolar spindle 1 (MPS1). The signal converter mitotic checkpoint complex (MCC) is an organization of MAD2, BUB3, BUB related 1 (BUBR1) and cell division cycle protein 20 (CDC20) A second organization is the ubiquitin ligase anaphase–promoting complex/cyclosome (APC/C) (78–80).
During prometaphase, the kinetochores of the chromosomes attach to the spindle microtubules (figure 7 A+B). The SAC is then active and monitors the state of kinetochore attachment. In case of improperly attached or unattached chromosomes the SAC signal is transduced. Subsequently, the SAC effector MCC, assembled at unattached kinetochores, binds and inhibits the APC/CCDC20-complex which regulates metaphase-anaphase transition. When all the chromosomes are properly attached to the mitotic spindle, the activation of the APC/CCDC20-complex enables Cyclin B and Securin ubiquitination and subsequent proteolysis leading to inactivation of Cyclin Dependent Kinase (CDK1) and activation of Separase. Inactivated CDK1 results in start of mitotic exit whereas active Separase cleaves the cohesin complex of the sister chromatids. This results in sister chromatid separation and finally to onset of anaphase. Moreover, other SAC key elements including MAD1, BUB1, MPS1, and Aurora-B increase the SAC signal and the amount of MCC assembly at the kinetochores (78, 79). Aurora-B is a serine/threonine (S/T) protein kinase functioning as a subunit of another complex named the chromosomal passenger complex which is discussed later.
Summary of Chapters
1. INTRODUCTION: Provides a background on assisted reproductive technologies (ART) and the challenges of chromosomal mosaicism in preimplantation embryos.
2. OBJECTIVES OF THE MASTER THESIS: Details the primary goal of establishing a simultaneous genome and transcriptome analysis protocol for single cells.
3. MATERIALS AND METHODS: Describes the experimental procedures, including single cell isolation, RNA/DNA separation, and various amplification and PCR techniques.
4. RESULTS: Presents the findings of the validation experiments for primer sets, magnetic bead separation, and sequencing data for single-cell samples.
5. DISCUSSION: Interprets the experimental outcomes, addressing amplification bias, cell-to-cell variability, and the effectiveness of the implemented protocol.
6. CONCLUSION: Summarizes the success of the implementation and outlines future applications for identifying genetic markers for embryo health.
Keywords
Single Cell, Genome, Transcriptome, Chromosomal Mosaicism, Preimplantation Genetic Testing, IVF, Embryogenesis, Mitosis, Spindle Assembly Checkpoint, Chromosomal Passenger Complex, Whole Genome Amplification, Sequencing, Aneuploidy, Gene Expression, hESC
Frequently Asked Questions
What is the primary focus of this research?
The work focuses on developing and validating a technique for simultaneous genome and transcriptome analysis at the single-cell level, specifically to study the origin of chromosomal abnormalities in human preimplantation embryos.
What are the central thematic areas?
The research covers reproductive genetics, assisted reproductive technology (ART), mitotic cell division mechanisms, and single-cell genomics and transcriptomics.
What is the main research question or objective?
The core objective is to determine if a shortage of specific maternal transcripts, such as those involved in the spindle assembly checkpoint (SAC) and chromosomal passenger complex (CPC), leads to an abnormal number of chromosomes in early embryonic development.
Which scientific methods are applied?
The study uses manual single-cell pick-up, magnetic bead-based physical separation of mRNA and gDNA, reverse transcription, multiple displacement amplification (MDA), Picoplex, qRT-PCR, and shallow whole genome sequencing.
What does the main part of the work cover?
The main section details the extensive Materials and Methods used to validate the protocol on fibroblasts and human embryonic stem cells, followed by a comprehensive Results section documenting the effectiveness of these methods.
Which keywords characterize the work?
Key terms include Single-Cell analysis, Preimplantation Genetic Testing, Chromosomal Mosaicism, Spindle Assembly Checkpoint, and simultaneous genome and transcriptome profiling.
Why is mRNA/gDNA separation considered a delicate step in this protocol?
It is the most critical step because incomplete transfer or cross-contamination between the mRNA (for the transcriptome) and gDNA (for the genome) can lead to noisy data, inconsistent copy-number profiles, or technical failure during sequencing.
How does this study address the limitations of bulk RNA analysis?
The study demonstrates that bulk analysis masks cell-to-cell variability in gene expression; by performing single-cell analysis, the researchers can reveal the inherent heterogeneity and transcriptional diversity that occurs during the early stages of cell division.
- Citar trabajo
- Marius Regin (Autor), 2018, Development of Simultaneous Genomic and Transcriptomic Analysis at the Single Cell Level, Múnich, GRIN Verlag, https://www.grin.com/document/490849
