Identification of immunological genes important for cytotoxicity

Official URL: http://risc01.sabanciuniv.edu/record=b2300597_ (Table of contents)

Natural Killer (NK) cells and Cytotoxic T lymphocytes (CTLs) are essential to defend the body against foreign or transformed cells. An understanding of the mechanism of their cytotoxicity can result in the generation of genetically modified cells with higher cytotoxicity and to identify drug-targets that increase cytotoxicity. In this thesis, genes which have a role in the cytotoxicity of CTLs were targeted by the CRISPR/Cas9 system to check whether they also play a role in the cytotoxicity of Natural Killer cells (NK-92 cell line). These cells are difficult to transfect with plasmid DNA and to generate single cell clones. In this thesis we attempted to optimize the Neon Electroporation method for the NK-92 cell line. Unfortunately, we could not obtain high transfection efficiency and high cell viability. We thus performed gene transfer into these cells by lentiviral infection. In order to obtain single cell cloned NK-92 cells, we generated a feeder cell line by transduction with lentivirus encoding the HSV-TK gene into the NK-92 cell line. Cell containing the TK gene can be negatively selected by ganciclovir treatment. We performed coculture experiments with this feeder line mixed with ganciclovir resistant, unmodified NK cells at different ratios under different doses of ganciclovir selection. We were able to initiate a healthy culture starting with only 17 NK-92 cells, using a 1:10000 ratio of ganciclovir sensitive feeder cells selected under increasing doses of ganciclovir from 0.01 μg/ml to 1 μg/ml. To mutate selected cytotoxicity genes, we expressed CRISPR/Cas9 by third generation lentivirus. Presence of a mutation and a decrease in mRNA expression level of the target genes was confirmed by the T7 assay and QRT-PCR. The cytotoxicity of mutated pools were assessed by a degranulation assay and by xCELLigence real time cell analysis. We identified several genes that are important for the cytotoxic response towards the MCF-7 cell line.