Epsilon Archive for Student Projects

Abstract

Background: The discipline of comparative oncology has advanced by recognising the importance of pet dog
spontaneous tumours as models for human cancer. Canine mammary tumours (CMTs) are pathologically and
molecularly similar to humans, with conserved oncogenic mechanisms.
Objective: This study aimed to analyse publicly available human breast cancer data sets and identify up and
downregulated genes, identify differently expressed gene in a mouse mammary tumour serglycin (SRGN)-deficient
environment, and to design and verify functioning stable CRISPR interference (CRISPRi) systems in the CMT-U27 cell
line, using two different plasmids containing dCas9-KRAB constructs, as the KRAB domain is a transcriptional
repressor known to downregulate wide range of genes, including transposable elements (TEs) and protein-coding genes
through epigenetic silencing. Then evaluate the interference of expression of SRGN, which is known to mediate
inflammation, remodelling of the extracellular matrix, and tumour progression.
Methods: A public transcriptomic dataset from mouse mammary tumours and four datasets from human breast cancer
samples were examined to study the expression profile of SRGN in the context of TP53-regulated genes.
Experimentally, CMT-U27 cells were transfected with dCas9-KRAB constructs and G418 was used for selection.
Subsequently, in the resulting G418-resistant cells qPCR verification was done to confirm construct expression and,
after addition of guide RNAs targeting exon 1 and exon 2, evaluating SRGN repression.
Results: Bioinformatics analyses uncovered that SRGN expression is variable in human datasets of breast cancer. In a
mouse knockout model, SRGN deficiency resulted in an extreme downward spiral of SRGN and inflammatory
extracellular matrix remodelling associated genes. CMT-U27 cells stably transfected with CRISPRi constructs were
shown to integrate the plasmid and exhibited variable SRGN repression. Most significant downregulation was seen in
clones CRISPI-G and CRISPI-G-2 with 0.70 and 0.39 fold changes, respectively. Non-targeting controls like CRISPI C
showed SRGN elevation, which may be explained by clonal heterogeneity after G418 selection.
Conclusion: The system for CRISPRi-dCas9-KRAB developed in this study should allow for efficient and precise
transcriptional control of genes of interest (GOI) in canine mammary cancer cells, extending beyond SRGN for future
gene suppression investigations. This system allows controlled exploration of manipulated genes, desired therapeutic
targets, and tumour biology in comparative oncology.