- Title
- Generation of gene disrupted mice to further elucidate the reproductive mechanisms of male factor fertility
- Creator
- Young, Samantha Allison Mary
- Relation
- University of Newcastle Research Higher Degree Thesis
- Resource Type
- thesis
- Date
- 2016
- Description
- Research Doctorate - Doctor of Philosophy (PhD)
- Description
- The issue of fertility is one of increasing importance in a global context, with infertility on the rise, along with increased use of assisted reproductive technologies. Yet our understanding of these often incredibly complex processes is still low, due in part to the difficulty in replicating the in vivo environment of fertilization in an in vitro setting, for any length of time. To counter this, we turn to genetically modified organisms, however, in the past the generation of animal models has been expensive, difficult and time consuming. The adaptation of the CRISPR/Cas9 system of genetic engineering to work in mammalian cells has allowed us to explore more than ever the role of certain genes in reproduction. One such animal model that is of particular use is the mouse. They are genetically similar to humans, allowing us to investigate conserved genes with potentially similar functions. They also have a relatively short reproductive cycle, making them effective for genetic modification, as several generations can be analysed within a short span of time. Herein, we examine the various uses of the CRISPR/Cas9 system in the mouse to target the specific area of male factor fertility. This system has developed dramatically in the few years that it has been available, from being able to generate a total gene knockout to subtler genetic modifications. We begin by utilizing the most basic function of the CRISPR/Cas9 system, generating a total gene knockout. Here, we describe for the first time the in vivo role of a gene previously thought to be linked to capacitation. The calcium-binding tyrosine phosphorylation-regulated protein (CABYR) is known in the literature to bind to the fibrous sheath of sperm flagella, however, by removing Cabyr by CRISPR/Cas9 gene knockout we observed that the role of CABYR is in fact in the correct formation of the fibrous sheath. This discovery adds much to the knowledge of the formation and function of the sperm flagella, perhaps even providing a diagnosis for male factor infertility due to poor sperm motility, which has previously been labelled as unexplained male factor infertility. This thesis also explores the use of CRISPR/Cas9 to delete regions of the genome, as well as performing amino acid substitutions. Whilst the genes chosen for these experiments led to no change in the fertility phenotype, it is the ability of the CRISPR/Cas9 system to perform these genetic modifications that is of great interest. The genes selected for this study were all involved in the formation or function of the dynein complex, the motor system that provides the flagella with the ability to move. The selection of these genes came from proteomic work that indicated phosphorylation changes, indicating a control mechanism for sperm motility. The fact that CRISPR/Cas9 could be used to substitute to potential phosphorylation sites demonstrates how much further we can take the investigation of fertility with the use of this system. Finally, we explored the use of the CRISPR/Cas9 system to insert a single nucleotide, creating a premature stop codon and truncation of the protein. The gene targeted was Izumo1. This gene is already known to be essential for fertilisation, yet with CRISPR/Cas9 we can study the individual domains of this, or other proteins in vivo. Taken together, the findings in this thesis demonstrate the many uses of the CRISPR/Cas9 system to investigate male factor fertility. This collection of studies explores its use from the basic, creating total gene knockouts, to the refined and subtle, amino acid substitution and insertion of a single nucleotide. The demonstration herein is of the potential of this system to address the specific needs of fertility research. Genes involved in fertility are often complex and backed up by several others to create significant redundancy. As shown with the CABYR knockout, in vitro investigation is not enough to give an accurate understanding of the role of any given gene. The advent of the CRISPR/Cas9 system with its simplicity, ease of use and speed renders it suitable as a tool for investigating the complex processes of male factor fertility.
- Subject
- CRISPR/Cas9; spermatozoa; mice; male factor fertility; knockout; thesis by publication
- Identifier
- http://hdl.handle.net/1959.13/1325823
- Identifier
- uon:25336
- Rights
- Copyright 2016 Samantha Allison Mary Young
- Language
- eng
- Full Text
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| Thumbnail | File | Description | Size | Format | |||
|---|---|---|---|---|---|---|---|
| View Details Download | ATTACHMENT01 | Thesis | 6 MB | Adobe Acrobat PDF | View Details Download | ||
| View Details Download | ATTACHMENT02 | Abstract | 354 KB | Adobe Acrobat PDF | View Details Download |