Epsilon Archive for Student Projects

Abstract

Serglycin is a proteoglycan expressed by many different cells, especially mast cells, but also by cancer cells. It has been reported that the level of expression of serglycin in cancer cells corresponds to the malignancy of the tumor. The higher level of expression the higher malignancy and poorer prognosis. Serglycin can promote migration, metastasis and epithelial to mesenchymal transition by binding to an adhesion molecule on the cells. However, up to this point very few, if any, studies have addressed the functional roles of serglycin in canine mastocytoma.
In this study, a canine mastocytoma cell line was cultured. Mastocytoma (MCT) is one of the most common cutaneous neoplasia in dogs accounting for up to 21% of all canine skin tumors. Apart from the cutaneous form there is also a systemic form which typically affects the liver, spleen, and bone marrow, although this form is uncommon. The cutaneous MCTs have a wide range in their manifestation. They can be a slow growing lesion mistaken for a non-neoplastic change or fast growing, ulcerated lesion with inflamed surrounding tissue.
To allow future explorations of the role of serglycin in carcinogenesis, this study was aimed to develop serglycin deficient canine mastocytoma cells using CRISPR/Cas9. At the site of the double strand break (DSB) by Cas9 a gene cassette was inserted, using electroporation, containing genes for antibiotic resistance and fluorescence in green and red. The DSB and the correct incorporation of the gene cassette was verified in a fluorescence light microscope, with PCR reactions and selection with antibiotics. In the fluorescence microscope it was discovered that the cells were autofluorescent in both green and red. The PCR reactions confirmed the 5´ side of the gene cassette to be present in the transfected cells. However, no results were given when amplifying the whole gene cassette or the 3´ side. This could indicate that the gene cassette was fractured or that the method was not successful. The conclusion of this study was that further analysis, for example subcloning and qPCR, is necessary to determine if the knockout of the serglycin gene was successful in the cells.