Establishment Of Cellular Senescence Model Using Lentiviral Vector Carrying Activated Oncogene

Oncogene activation is a key step in tumorigenesis. Expression of theoncogenic form of RAS transforms rodent cells efficiently when expressed incombination with other oncogenes. However, its sole expression resultedparadoxically in an irreversible arrest in a range of mammalian cell types.Serrano reported their observation on Cell magazine in 1997.this was the firstreport of the phenomenon called oncogene induced senescence (OIS). Cellularsenescence probably suppresses tumorigenesis because cancer developmentrequirescellproliferation.OIS was first identified in cultured cells and does not occur in all cells, sois it physiologically relevant? In 2005, the significance of OIS in vivo was demonstrated by several independent reports. Indeed, the discovery thatcommon human naevi are constituted of senescent melanocytes carryingBRAF-activating mutations makes the phenomenon of cellular senescence acommonphysiologicaloccurrence.In this study, we repeated the experiment of Serrano in 1997,Establishedthe classic model of OIS. Activated oncogenes were transduced into fibroblastthrough lentiviral vectors instead of traditional retroviral vectors. Theexperimentscanbedividedintothreeparts:1. Establishment of cellular Senescence model using hydrogen peroxide andcisplatin respectively. Hydrogen peroxide represents Oxidative stress and cisplatin DNAdamage stress, both of the two drugs have been reported to have the ability of inducingcellular senescence. Based on former reports, we established an easier and faster model ofcellularsenescence.2. Reconstruction oflentiviralvector. Once we decided to use the lentivirusas the toolforgenetransfer,weorderedlentiviral packagingsystems from Addgene website.Inorder to make stable antibiotic selection after lentiviral infection, we reconstructed thelentiviralvector. In this part we demonstrated that theresultingconstructionscouldbestableselectedbyantibiotic.3. Establishment of cellular Senescence model using lentiviral vector carryingactivated oncogene. Based on the lentiviral vector we constructed in part two, in this partwe successfully constructed lentiviral vector containing H-rasV12 and c-myc respectively.Lentiviral particles were packaged and target cells were infected, followed byappropriate antibiotic selection. We found that the lentiviral particles carryingH-rasV12 have successfully induced cellular senescence reponse, but the effectoflentiviralparticlescarryingc-mycneedsfurtherinvestigation.