Zweekhorst, Wyco (2024) Genomic engineering at the Roco4 & mybW locus in Dictyostelium discoideum using CRISPR/Cas9. Scientific Research Project, Science Education and Communication.
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Abstract
Dictyostelium is an important eukaryotic model organism for research into basic cell biological mechanisms. It can be used to study systems such as growth, micropinocytosis, cell mortality, chemotaxis, and signal transduction during development. Recently, CRISPR/Cas9 has been added to the toolbox for functional analysis in Dictyostelium, which makes it possible to modify genomes at a higher efficiency. The aim of this project was to implement the CRISPR/Cas9 system for Dictyostelium in the cell biochemistry lab using Roco4 and mybW as the model genes. To achieve this goal, Roco4 and mybW knockouts and GFP-inserts were generated using the CRISPR/Cas9 system. Post development of the knockouts and GFP-inserts, the Roco4 knockouts and GFP-inserts served to give insight into the Role of Roco4 during phagocytosis. The main questions addressed whether it was possible to visualize Rab protein phosphorylation by Roco4 during phagocytosis and if Roco4 plays a role in phagosomal uptake and maturation. In the end, no successful CRISPR/Cas9 knockouts or GFP-inserts were generated for either Roco4 or mybW. Consequently, the assays required to answer the questions surrounding the role of Roco4 in phagocytosis were performed using a pre-existing Roco4 null cell line. Through the use of an immunofluorescence assay utilizing the pT72-Rab8a antibody, this project successfully visualized the phosphorylation of Rab proteins by Roco4 in Dictyostelium cells.
| Item Type: | Thesis (Scientific Research Project) |
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| Supervisor name: | Kortholt, A. and Bianchi, F. |
| Degree programme: | Science Education and Communication |
| Thesis type: | Scientific Research Project |
| Language: | English |
| Date Deposited: | 14 May 2024 09:29 |
| Last Modified: | 14 May 2024 09:29 |
| URI: | https://fse.studenttheses.ub.rug.nl/id/eprint/32393 |
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