Generation of Apak stably knockout cell lines and variation of their p53 activity and apoptosis / 军事医学

ABSTRACT

Objective To establish an Apak gene stable and permanent knockout cell line using CRISPR/Cas9 system in human colon cancer cells ( HCT116 cells), and study the effect of Apak knock-out on p53 activity and apoptosis. Methods The lentiCRISPR v2-sgRNA Apak expression plasmid was co-transfected with lentivirus coated plasmids pSPAX2 and pMD2.G.The supernatant was collected, filtered, and used to infect HCT116 cells.The positive clones were screened out by puromycin culture and Western blot was used to detect Apak knockout cell lines.Luciferase reporter gene assay, flow cytometry analysis and colony formation assay were used to examine p53 activity and apoptosis of Apak knockout cells, respectively.Results Apak knockout HCT116 cell lines were generated in which p53 activity and apoptosis were increased,but the colony formation was decreased.Conclusion The Apak stable knockout cell lines of HCT116 are successfully generated by CRISPR/Cas9 system for further functional study.