Epidermolysis Bullosa simplex (EBS) is a rare genetic disorder that is typically inherited
in an autosomal dominant fashion and affects approximately 1 out of 20 000 individuals. This
disease is caused by mutations in either the KRT14, KRT5 or PLEC genes. These genes code for
proteins involved in the formation of the cytoskeleton in basal keratinocytes, which form the
basal layer of the epidermis. The cytoskeleton provides structural support to the basal
keratinocytes and mutations in these genes cause cytoskeletal malformations, making these cells
more susceptible to physical stress. This results in the cells undergoing lysis under trivial
mechanical stress and causing the epidermis to detach from the dermis, the layer immediately
below the epidermis. This leads to the primary symptom of EBS: the formation of blisters. The
goal of this project is to recreate EBS in zebrafish using transgenesis and to create stable mutant
transgenic line. In the future, high throughput drug screening will be done on mutant zebrafish
embryos to find potential drug candidates that can alleviate the symptoms of EBS. To
accomplish this, missense and deletion mutations in zebrafish krt5 cDNA using site-directed
mutagenesis were performed. It was previously shown that mice models for this disease die
shortly after birth and thus no stable mutant lines were able to be created. To ensure embryo
survival and avoid a similar fate, mutant krt5 cDNA was expressed in non-essential tissue, such
as the embryonic fin fold using a fin epithelial-specific enhancer named epi. These constructs
were injected into one-cell stage zebrafish embryos, which were raised and screened for
integration of the construct in their germ cells. While results from injected embryos were
promising, mutant transgenic zebrafish did not demonstrate any blistering. In an attempt to
induce blistering, mutant zebrafish embryos were placed under various environmental stressors
known to worsen the symptoms of EBS. This was not successful. Expression of mutant keratin 5
in the basal epidermis of the entire embryo using the 2.3kb upstream region of the zebrafish krt5
gene to drive expression also did not yield any results. More investigations are needed to
determine if it will be possible to use the zebrafish to model EBS.
| Identifer | oai:union.ndltd.org:uottawa.ca/oai:ruor.uottawa.ca:10393/38249 |
| Date | 09 October 2018 |
| Creators | MacDonnell, Samuel |
| Contributors | Akimenko, Marie-Andrée |
| Publisher | Université d'Ottawa / University of Ottawa |
| Source Sets | Université d’Ottawa |
| Language | English |
| Detected Language | English |
| Type | Thesis |
| Format | application/pdf |
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