Protein degradation via the ubiquitin-proteasome system is a key cellular mechanism that regulates protein activity. Specificity of this pathway is granted by an F-box protein, and to date, more than 70 F-box proteins have been found within mouse and human genomes. A number of critical pathways, such as cell growth/survival/death are regulated by F-box proteins and mutations in these proteins are often implicated in cancers. Around 10 to15% of colorectal cancers share a mutation in a common F-box protein. Genetic studies of these proteins are incomplete owing to the size of this protein family and in vivo studies are not always possible due to embryonic lethality of knockout mutations.
Why we funded it
This PhD Studentship aims to replace genetically modified (GM) mice for the study of F-box proteins with 3D in vitro intestinal organoid cultures. Relevant mutations will be introduced with the CRISPR/Cas9 genome editing system.
The generation of GM mice containing relevant homozygous mutations requires approximately 400 to 500 mice per year. Dr Shams Nateri predicts a further 400 to 500 mice are required for functional experiments to determine the effect of the removal of the F-box protein of interest. The use of in vitro organoid models and genome editing systems has the potential to replace all the mice needed for these studies.
3D intestinal organoids replicate gut physiology more faithfully compared to 2D monolayer stem cell models as stem cells cannot differentiate into fully mature intestinal epithelial cells. A novel fluorescent/luminescent-based screening system will be employed for the creation of knockout organoids. The use of this system will allow for the creation of a large scale organoid library with targeted editing of F-box proteins. Any phenotypes derived from this library can be fully explored and the attributed mutation identified. The increased complexity of the 3D intestinal organoid, compared to 2D models, also allows for the model to be potentially used in drug screening studies.
Hossein Kashfi, S. et al. (2018). Morphological alterations of cultured human colorectal matched tumour and healthy organoids. Oncotarget 9(12): 10572-10584 doi: 10.18632/oncotarget.24279