Epsilon Archive for Student Projects

Abstract

Cancer is one of the most common causes of death amongst dogs, where visceral hemangiosarcoma is one of the most aggressive and life threatening. Research has shown that early detection is key to a better prognosis and that the proteoglycan serglycin can hold valuable information. In this thesis a serglycin defective canine hemangiosarcoma cell line was developed, using the gene editing method CRISPR/Cas9, allowing for a detailed functional investigation of serglycin in this aggressive cancer form.
Serglycin is a proteoglycan strongly associated with inflammation and the cells regulatory system in different immune cells. It’s a protein linked to various forms of cancer, and it has been associated with increased malignancy. Here we lipofectamine transfected the canine hemangiosarcoma cell line DD-1 and the human angiosarcoma cell line ISO-HAS-B with the CRISPR/Cas9 + guideRNA nucleoprotein complex and a donor cassette giving cells green and red fluorescence as well as antibiotic-resistant abilities. Transfection was followed by FACS single cell sorting to create sub clones, followed by genomic PCR and control sequencing of the target site. Clones with
changes in the target site sequence were then tested for serglycin expression using qPCR. Due to the resemblance between canine hemangiosarcoma and human angiosarcoma cancer forms we also aim to perform a comparative analysis of the two cell lines.
Results show that transfecting cells with lipofectamine is an effective and productive way to insert the donor DNA cassette in both the DD-1 and the ISO-HAS-B cell line. Transfected cells from both cell lines were found to be emitting green, red and some cells emitting a double fluorescence indicating a bi-allelic knockout. However, while DD-1 was growing well after single cell sorting,
ISO-HAS-B rapidly went into a growth arrest when single cell cultured. Sequencing of the PCR product spanning the desired target site in the genome indicated non homologous end joining (NHEJ) in three out of four canine DD-1 cell line clones, and when analyzed by qPCR two subclones were found to no longer express serglycin.
With that we conclude that we might have a serglycin defect canine hemangiosarcoma cell line.