The utilization of CRISPR-Cas9 to establish novel ?X?2 knockout cell lines in mice



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The ?X?2 integrin functions as an important mediator of the complement system, and undergoes inactive to active state by switching its conformation from bent closed to extended open, contingent on binding of different ligands. This extended open conformation has higher affinity for cognate ligands including iC3B, an important molecule of the complement cascade. Thus, the extended open conformation leads to an inflammatory response, and the bent closed conformation suppresses inflammation. Despite the importance of this integrin in mediating the inflammatory response of the human immune system, there have not been studies of the ?X?2 integrin locked in alternate conformational states in a mouse model due to the lack of cell lines. My project has been to create Cas9-plasmid to knock out ?X and ?2 genes from any mouse cell line. By designing specific oligonucleotides that Cas9 can utilize to make double stranded cuts in the genes for ?X and ?2, novel cell lines can be created from wild-type DC2.4 and RAW264.7 mouse cells through transfection. These novel cell lines can then be used for future research, such as how the ?X?2 integrin in an alternate state can affect the pathophysiology of diseases such as cancer, inflammatory and autoimmune diseases.