NLRP INFLAMMASOME : CRISPR knockout of RIPK3 gene in THP-1 monocytes

Abstract: Inflammation is a biological response to harmful stimuli which assists the overall immune response in defending the organism against potential threats like bacteria and other pathogens. This response is largely regulated by multiprotein complexes known as inflammasomes, which use one or more multi-stage networks to create a coordinated response across cell groups. The NLRP3 inflammasome in particular is thought to have many interacting partners in a complex inflammation and cell-death management network. RIPK3 is one protein that has been implicated in regulation of inflammation and cell death pathways in connection with the NLRP3 inflammasome. In this study, we attempt to create a CRISPR/CAS9 construct to knockout the RIPK3 gene in THP-1-ASC-GFP monocytes with ViafectTM Transfection in order to examine the apparent effects at various stages of activation. Transfected cells were then quantitatively examined using qPCR. While the Guide-itTM CRISPR/CAS9 Systems kit produced high quantity, high quality DNA plasmids, this study found that the ViafectTM system resulted in reduced RNA isolation among cells differentiated with PMA as compared to non-differentiated counterparts. qPCR performed on cDNA generated from the RNA extractions resulted in highly erratic Cq values with standard deviations well above the acceptable limitations among technical replicates in both experimental and control gene samples. Additionally, this study found that the reference gene used, ACTB, did not maintain stability across samples. Due to mistakes and time constraints, the experiment failed to provide any substantial evidence of activity; however, an architecture is developed for optimization of future studies.