Experimental gene expression modulation - A practical and theoretical approach

Abstract

The aim of this project was to create a stable knockout cell model with the CRISPR/Cas9 method for programmable genome editing that would make it possible to run long term assays to further investigate the cellular function of the long non-coding RNA FAM83H-AS1. The CRISPR/Cas9 method is compared to gene knockdown with small interfering RNAs (siRNAs) and short hairpin RNAs (shRNAs) via the RNA interference (RNAi) system in cells. The cell lines T-47D, MDA-MB-468, and BT474 were transfected with the CRISPR/Cas9 components by electroporation and single cell sorted by flow cytometry. Wild type T-47D cells were transfected with siRNAs towards the target gene. knockout status of the CRISPR clones was determined by PCR, and the expression levels of FAM83H-AS1 and FAM83H was measured with RT-qPCR. Our attempt at creating a CRISPR knockout cell line resulted in eight T-47D CRISPR clones; three heterozygous knockout clones, and five clones where the FAM83H-AS1 gene remained intact in both alleles. We were not successful at making a homozygous knockout clone, or expanding CRISPR clones in MDA-MB-468 and BT474 cells. Analyses of the T-47D clones with RT-qPCR showed variable expression levels of FAM83H-AS1 in the three heterozygous clones. The expression level of FAM83H seems to follow the expression of FAM83H-AS1 in all clones. This was not seen with siRNA knockdown of FAM83H-AS1, and suggests a relationship between the sense, FAM83H, and antisense gene, FAM83H-AS1, at the transcriptional level. Conclusively, there are several advantages and disadvantages with each knockdown strategy, and as our results show the CRISPR/Cas9 method is not the most suitable option for long term knockout of gene expression in our epithelial cell lines. Knockdown with shRNA might prove to be a feasible alternative as stable gene silencing is possible and some of the methodological issues with the CRISPR/Cas9 technique is overcome.