Possible proliferation control of mammalian cells by selective disruption of signaling through the MAPK-pathway
Independent thesis Advanced level (degree of Master (Two Years)), 20 credits / 30 HE creditsStudent thesis
In this study we deliver genes coding for chimeric proteins consisting of an affinity ligand (Affibody molecule; binding to Ras or Raf) attached to a degrading signal through a lentiviral delivery system. The goal is to degrade Ras and Raf inside the cell to create a system that allows for control of proliferation through inhibition of signaling through the MAPK signal pathway. Three different constructs were analyzed utilizing slightly different ways to degrade Ras or Raf proteins through the endogenous proteasomal degradation system. The first construct emphasize on using the proteasomes own degradation tag ubiquitin attached to the Affibody molecule. The second uses Protax technology destilled from HIF1 to recruit the Affibody molecule. The second uses Protac technology destilled from HIF1 to recruit the Afribody molecules to the von Hippel Lindau (VHL) part of the VBC-Cul2 E3 ubiquitin ligase that actively marks HIF1 for proteasomal destruction under normoxic contitions. Last but not least a construct utilizing the TNFα inducible IKBα protein that is degraded as a stress response to TNFα stimulation in cells. As a control we utilized the Affibody molecule ZTaq which was not expected to interact with any protein inside the cell. Within the IKBα track an additional construct was also assembled that contained an affinity matured variant of the Affibody binding to Raf, henceforth called Raf3. Delivery of genes were successful and the cells were monitored closely. Proliferation stalled slightly but, after extensive testing, no solid conclusions could be made if the proposed effect was achieved or simply toxic for the cells. Extended testing is needed to make any decisions on the future of the concept.
Place, publisher, year, edition, pages
Engineering and Technology
IdentifiersURN: urn:nbn:se:kth:diva-149651OAI: oai:DiVA.org:kth-149651DiVA: diva2:744697