Epsilon Archive for Student Projects

Abstract

Canine mammary tumor (CMT) is the most common form of cancer in bitches and is a common cause of death. Around a third to a half of the CMT are malignant, and carcinomas are a majority of those. In this study a cell line, called CMT-U27, derived from a canine mammary carcinoma was subjected to an attempt to knock out a gene by using the common gene editing method CRISPR/Cas9. With CRISPR/Cas9 it is possible to create a double-strand break in the DNA and insert a piece of donor DNA to give the cell certain desired abilities. In this study the donor DNA used contained sequences for fluorescent proteins and antibiotic resistances to enable the selection of the cells with a successful knock-out.
The goal was to knock out the gene for serglycin, a proteoglycan expressed by a wide variety of cells. Serglycin is strongly associated with inflammation and has normally many different functions. Among them, it has an important role in storage and secretion of bioactive substances in different immune cells and plays a part in regulation of inflammation. Changes in the expression of serglycin has been reported in multiple forms of cancers and serglycin has been linked to an increased malignancy. Establishing a serglycin-deficient cell line would facilitate further research about the involvement of serglycin in carcinogenesis.
The CMT-U27 cell line were transfected with the donor DNA by using the method electroporation. The cells then underwent an antibiotic selection to try to discern the transfected cells from the others.
The cells showed promising results early after the transfection. Some of the cells emitted green and/or red fluorescence which is a sign that the donor DNA have been incorporated and expressed. A double fluorescence can indicate a bi-allelic knock-out which is the desired outcome. Unfortuna-tely, the cells did not survive the antibiotic selection. A possible reason for their death could be an unsuccessful integration of the donor at the desired location or a too immediate start of the antibiotic selection after the transfection. No confirmed serglycin-deficient cell line was able to be established due to a lack of time but experiences from this experiment will be valuable for future trials.